RBM20 gene variants associated with left atrial dilatation in patients with old myocardial infarction and heart failure with reduced ejection fraction genotype-phenotype relationships of

Aim. To study the prevalence of RBM20 gene polymorphisms and their relationship with the structural and func tional left atrial (LA) characteristics in patients with coronary artery disease and heart failure with reduced ejection fraction (HFrEF). Material and methods. The study included 138 men aged 55,8±6,6 years with prior myocardial infarction ≥ 12 months ago and HFrEF (class II-IV heart failure, left ventricular ejection fraction (Simpson’s methods), 25,1±7,2%). The cont rol group consisted of 384 healthy donors. Genotyping of two RBM20 polymorphic variants (rs942077 and rs35141404) was performed by real-time polymerase chain reaction. Results. The prevalence of RBM20 polymorphisms did not dif fer in the HFrEF cohort and the control group. The GA rs35141404 genotype was more common among patients with a less pronounced increase in LA volume index (LAVI) (p=0,034). The minor A allele rs35141404 was associated with a protective effect on severe LA remodeling. However, this association did not reach the level of significance. Conclusion. For the rs942077 and rs35141404 polymorphic variants of the RBM20 gene, no significant associations were found with the LA size and atrial fibrillation presence in patients with HFrEF and old myocardial infarction. There was a tendency towards the association of the A allele and the GA rs35141404 genotype with a protective effect on LA remodeling. The data obtained confirm the need for further search for a wider po pulation of patients with heart failure and coronary artery disease.

The influence of polymorphic genetic variants on development and course of various cardiovascular diseases has been noted and actively studied for many years, but the volume of study in this area has increased significantly after development of new me thods of molecular biology and genetics. The ap plication of genome-wide studies and the use of next-generation sequencing allowed to detect new determinants associated with the risk of development and adverse outcome of chronic heart failure (CHF) of various etiologies. At the same time, the influence of genetic factors on some structural and functional parameters of the cardiac muscle in CHF development and myocardial remodeling remained poorly studied.
In recent years, many studies have been published analyzing the association of various genetic deter mi nants with myocardial structural para meters and cardiac chamber sizes. The greatest number of works was devoted to the search of geno type-phenotypic associations in development of patho logical myocardial remodeling of predomi nantly dilated phe notype. At the same time, single works were devo ted to the analysis of genetic variants associated with structural atrial remodeling. Thus, in 2010, the rela tionship between genetic variants of polymorphic loci in the genes NTN1, MYH10, COX10 and MYOCD with left atrium (LA) size was demonstrated [1]. In 2015, Mints Y, et al. confirmed the association of the polymorphism rs10033464, located near the gene PITX2 in locus 4q25, with LA dilatation development [2]. The gene PITX2 encodes the protein Pitx2, which is a transcription factor that is a member of the RIEG/Pitx family of homeodomain transcription factors that play an essential role in embryonic development. It is important to note that this transcription factor undergoes alternative splicing, resulting in four isoforms (Pitx2A, Pitx2B, Pitx2C, PitxD) in the body and is connected to LA formation and struc tural and morphological features of pulmonary venous mouths [3]. This transcription factor is expres sed asymmetrically during different stages of cardiac development and plays an integral role in cardiogenesis, and insufficiency of its function leads to development of structural and electrophysiological abnormalities in atrium [4].
Another foreign study demonstrated the association of the above-described polymorphism rs10033464 with early recurrence of atrial fibrillation (AF) after cardioversion and larger LV size in a meta-analysis of 7034 patients with this rhythm disorder [5]. The mechanism of structural and functional interconnections of the LA tissue remains incompletely understood. Atrium dilation is thought to cause activation of signal-dependent extracellular kinase Erk1/ERk2, causing further progression of atrium tissue fibrosis, formation of re-entry foci and contributing to arrhythmogenesis [6]. There is also an inverse relationship: LA dilation can oc cur due to AF in patients without its primary expansion. Currently, the search for genotype-pheno typic asso ciations with regard to structural and functio nal remodeling of cardiac chambers is actively continuing [5,6].
The development of dilatational remodeling is asso ciated with multiple genetic determinants of myocardial structural proteins, including the gene RBM20, pathogenic variants in which lead to develop ment of arrhythmogenic cardiomyopathy in ag gressive form [7]. The gene RBM20 encodes the protein RBM20, which is a transcriptional splicing factor for many genes expressed in cardiac muscle and involved in maintaining sarcomere structure, diastolic function and ion transport, such as: TTN, CaMKII, CACNA1C, LDB3, LMO7, FHOD3, PDLIM3, RTN4, TRDN, OBSCN, RYR2 [7,8]. The main target of RBM20 is the gene TTN encoding the giant protein taytin, which acts as a spring that provides sarcomere stiffness and plays an important role in passive relaxation of the cardiomyocyte [9]. It is noteworthy that 2 above-mentioned genes, TTN and RBM20, are predominantly associated with the development of dilated cardiomyopathy (DCM), and in the last 3 years much attention has been paid to studying the role of their genetic variants in development of arrhythmic events in patients with CHF and DCM, including AF, both in inherited forms of cardiomyopathies and in the general population [1,9].
It seems relevant to study the relationship of gene tic determinants, such as polymorphic variants of the gene RBM20 (rs942077 and rs35141404), with struc tural and functional characteristics of LA in patients with heart failure (HF) with low ejection fraction (HFrEF) of predominantly non-monogenic nature.

Material and methods
This study is a single-center study including 138 men with coronary heart disease, postinfarction cardiosclerosis, and stable HFrEF of II-IV functional class, aged 40-68 years, on standard drug therapy, who were treated at the V. A. Almazov Federal State Medical Research Center in 2012-2018. The control group was represented by practically healthy people who constituted the donor control base of the V. A. Almazov Scientific Research Center (384 people), comparable in age to the studied cohort. The criteria for non-inclusion in the study were: primary and postmyocardial DCM, hypertrophic cardiomyopathy, hemodynamically significant organic heart valve lesions, cardiac chamber dilata-tion due to accumulation disease, secondary arterial hypertension (AH), extensive cardiac surgery or per cutaneous coronary intervention or valvuloplasty, and electrophysiology intervention within 12 months before randomization, acute or decompensated HF less than 3 months prior to inclusion.
The study was approved by the ethical committee of the V. A. Almazov Scientific Research Center and was conducted in accordance with good clinical practice and the ethical standards of the Declaration of Helsinki. All respondents signed an informed consent for the necessary examination methods.
Patients' status was assessed, and routine laboratory and instrumental diagnostic methods were performed. Echocardiography was performed accor ding to the standard protocol of the center. The LA volume (LAV) was indexed to the body surface area (BSA) and the degree of severity of growth (in meters). Normative values of the LA size: LAV (ml)/BSA (m 2 ) and LAV (ml)/(height, m) 2 specific for gender was determined in accordance with the recommendations of the American and European associations of echocardiographic (ASE/ EAE) -2015 for echocardiography in adults and the European communities on cardiology and hypertension (ESC/ESH) -2018 for treatment of hypertensive patients (≤40 mm, ≤34 ml/BSA (m) 2 and ≤18,5 ml/(height in m) 2 , respectively) [10,11]. The ejection fraction (EF) of the left ventricle (LV) was calculated using the Simpson method. The study included patients with LV EF <35%.
Characteristics of patients. The main characteristics of the cohort under study are given in Table 1. The average age of patients was 55,8±6,6 years. Most respondents had undergone myocardial revascularization and had HFrEF of functional class II. AH, diabetes mellitus and obesity were observed in 96%, 17%, and 23% of cases, respectively. Implanted devices, including permanent pacemaker, implanted cardioverter defibrillator, cardiac resynchronization therapy device, cardiac contractility modulator had 60% of patients. According to the anamnesis and programming results, AF (constant, persistent or paro xysmal form) was registered in 27% of cases. LV EF was 25,1±7,2%. An increase in LA volumes was detected in more than 90% of cases.
To study polymorphic variants of RBM20 (rs942077 and rs35141404), DNA was isolated from whole blood using the FlexiGene DNA Kit (Catalog No.51206). These polymorphic variants were identified by realtime polymerase chain reaction using allele-specific primers from Applied Biosystems on an Applied Biosystems 7500 RealTimePCRSystem amplifier and Syntol reagent kit.
Statistical processing was performed using the Sta tistica 10 package. Data are presented as: mean ± standard deviation (M±SD) in case of normal distribution, medians (Me) and 25% (Q25) and 75% (Q75) quartiles, frequencies, and percentages of the total number of follow-up n (%). Continuous values with a normal distribution were compared with each other using a t-test. The nonparametric Mann-Whitney test for independent samples was used to compare continuous values with a distribution other than normal. Independent categorical data were compared using Fisher's two-sided test and chisquare (χ 2 ) test with Yates continuity correction at statistical significance p<0,05.

Results
The occurrence of alleles and genotypes of the polymorphic variants of RBM20 (rs942077 and rs35141404) in the presented sample of patients with HFrEF did not differ compared with the control group (Table 2). There were also no differences in Table 1 Characteristics of patients the prevalence of the studied RBM20 gene polymorphisms depending on the presence of AF (Table 3). Given the high detectability of LA dilatation in this cohort of patients, the analysis of the relationship between SNP rs942077 and rs35141404 of the gene RBM20 with the degree of gender-specific increase in LA size according to ASE/EAE recommendations was carried out [11]. As a result of this analysis, no statistically significant difference in the frequency of genotypes and alleles of RBM20 polymorphic variants was obtained (Table 4).
It is known that among different parameters of LA there is more prognostic value with regard to adverse cardiovascular outcomes in the index of LAV/BSA regardless of the presence of AF, HF and its etiology [12]. The values of LAV index (LAVI) of >50 ml/m 2 and >40 ml/m 2 had the same predictive ability with LV EF in relation to hospitalizations due to HF and mortality in patients with coronary heart disease [13]. Therefore, we attempted to analyze the prevalence of the studied RBM20 SNPs in patients with varying degrees of LAVI enlargement.
When analyzing alleles and genotypes rs942077 and rs35141404 RBM20 in the study groups, depending on the presence of severe LA dilatation according to LAVI indicator, significant differences in the representation of the GA genotype rs35141404 were detected. In patients with LAVI <40 ml/m 2 , cor responding to less pronounced LA dilatation according to ASE/EAE classification, was dominated by GA genotype and allele A rs35141404 compared with patients with LAVI 50 ml/m 2 (38%:16%, p=0,034% and 19%:11%, p>0,05). At the same time,  there was a higher prevalence of GG genotype among patient groups with LAVI ≥40 ml/m 2 and >50 ml/m 2 when the threshold of significance was not reached (79%:62% and 81%:62%, respectively, all p>0,05). Patients with LAVI <40 ml/m 2 had less pronounced clinical manifestations of CHF and greater LV EF compared with patients in the reference group with LAVI >50 ml/m 2 . AF was observed more frequently among the contingent with more pronounced LA dilatation (p>0,05). Having said so, the subgroups of patients did not differ in age, body mass index, AH frequency and its duration, prevalence of diabetes mellitus, obesity (all p>0,05). Data on subgroups of patients are given in Tables 5 and 6.

Discussion
Currently, much attention is paid to the study of molecular determinants associated with the develop ment of various HF phenotypes and myo cardial remodeling, the so-called molecular epi demiology of CHF, in which an important place is given to genetic predictors [14].
The gene RBM20 regulates the splicing of many cytoskeletal protein genes, including TTN, CAMK2D, LDB3, LMO7, PDLIM3, RTN4, and RYR2, and is also connected to sarcomere assembly, ion transport and posttranslational splicing of a number of calcium signaling and calcium homeostasis protein genes [7]. The listed functions of the gene RBM20 largely Table 4 Distribution of genotypes and alleles of polymorphic variants rs942077 and rs35141404 of the gene RBM20 in groups of patients with HFrEF depending on the size of LA  determine the high frequency of arrhythmological events in patients carrying pathogenic variants of RBM20 [1]. In this regard, it can be assumed the presence of genotype-phenotypic associations of polymorphic variants of this gene with regard to structural remodeling, in particular, of atrium tissue in CHF of noninherited genesis. In our study, the occurrence of genotypes and alleles of polymorphic variants rs942077 and rs35141404 of the gene RBM20 in patients in the narrow phenotypic HFrEF group with postinfarct cardio sclerosis did not differ from data obtained in controls and dbSNP genetic database data, but SNP rs35141404 was associated with LA dilatation. The GA rs35141404 genotype was significantly more com mon in patients with a less pronounced increase in LAVI. A similar association was seen for the preva lence of minor allele A (rs35141404), although the signi ficance threshold was not reached. It is impor tant to note that the group of patients with the highest occurrence of this genotype and allele did not differ in age and cardiometabolic factors contributing to LA dilatation from the reference group with more pronounced LA remodeling and, naturally, a higher incidence of AF, which largely agrees with the work of Refaat MM, et al. (2012) [15]. When studying the associations of SNP rs942077 and rs35141404 of the gene RBM20 with arrhythmic events and outcomes in a sample of patients with HFrEF of coronary etio logy and DCM, the authors demonstrated the association of rs35141404 polymorphism of the gene RBM20 with AF regardless of the CHF causative factor. It is worth noting that in this work, similar to the data we obtained, it was the rs35141404 A allele that was associated with a protective effect in relation to the risk of developing AF (odds ratio 0,59, 95% confidence interval 0,40-0,84, p=0,006) [15].
The results of several recent studies demonstrate the association of rare, shortening variants of the gene TTN (TTNtv) with AF, CHF and lower LV EF in patients even without the diagnosis of DCM [16]. Taking into account these data and the key role of RBM20 in the TTN splicing process, further inves tigation of the molecular mechanisms of pathogenesis and structural and functional myo cardial remo deling in CHF of ischemic etiology associated with RBM20 and shortening variants and/or pathogenic SNPs of the tetin gene seems relevant, providing a potential opportunity to develop a personalized medical approach.
Study limitations. An important limitation of this study was the relatively small sample size of the study group (138 patients), the presence of possible additional causal factors potentially influencing myocardial remodeling, including the ongoing drug and non-drug therapy, which may have an effect on the LA dilatation development. Only male patients were included in the work, while the control group had no gender restrictions. The above predetermines the cohort expansion of the studied patient population and the application of multivariate statistical methods for a more complete understanding of genotypephenotypic associations.

Conclusion
The polymorphic variant and rs35141404 of the gene RBM20 is associated with the severity of LA dilatation in patients with HFrEF and pronounced postinfarction myocardial remodeling. Our preli- Table 6 Clinical characteristics of groups of patients with HFrEF depending on LAVI minary data support the need to further search for genotype-phenotypic associations in the broader population of patients with CHF of coronary etiology in the focus of personalized medicine.
Relationships and Activities. The study was performed at the expense and within the framework of the state assignment of the V. A. Almazov Scientific Research Center Reg. No. АААА19-119070490034-4.