The relationship between serum miR-21 levels and left atrium dilation in elderly patients with essential hypertension

Yan WANG, Guang-Ping FU, Qiu-Yan WANG Ting-Ting ZHANG Li-Li HE Qing-Juan ZUO Chang-Lei ZHANG, Yi-Fang GUO

1. Department of Geriatric Cardiology, Hebei General Hospital, Shijiazhuang, Hebei, China; 2. Hebei Key Laboratory of Forensic Medicine, Department of Forensic Medical, Hebei Medical University, Shijiazhuang, Hebei, China; 3. Hebei Medical University, Shijiazhuang, Hebei, China

ABSTRACT BACKGROUND MicroRNA-21 (miR-21) is related to hypertension and cardiac remodelling. Left atrium (LA) dilation is highly sensitive to small haemodynamic changes in the left ventricle (LV) that are induced by hypertension. This study aimed to elucidate the relationship between miR-21 expression and LA dilation in elderly patients with essential hypertension (EH). METHODS In this cross-sectional study, one hundred elderly patients with EH were recruited for the study. According to their left atrium diameters (LADs), the patients were divided into the LA dilation group [42 patients (42%)] and the no-LA dilation group [58 patients (58%)]. The serum levels of miR-21 and chemical biomarkers used in the clinic, such as creatinine, blood urea nitrogen, uric acid, fasting blood glucose, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol, Lp(a), apolipoprotein A1 (apoA1), and apolipoprotein B, were measured. All the patients underwent echocardiographic examination, and the LAD, interventricular septum (IVS), right atrium diameter (RAD), right ventricle diameter (RVD), left ventricular end-systolic diameter (LVESD), left ventricular end-systolic diameter (LVEDD) and left ventricular ejection fraction (LVEF) were measured. RESULTS The levels of miR-21 [8.02 (5.21, 14.39) vs. 6.05 (3.81, 8.95), P = 0.011] and LVEF (67.02 ± 3.82 vs. 64.14 ± 4.43, P =0.001) were higher in the LA dilation group. The levels of creatinine [70.40 (64.45, 80.15) vs. 63.9(60.1, 73.43)], P = 0.020] were higher in the no-LA dilation group. The levels of HDLC (r = - 0.209, P = 0.037), apoA1 (r = -0.269, P = 0.007) and RAD (r = 0.203, P =0.043) were significantly correlated with miR-21 expression. The LAD was significantly correlated with the RAD (r = 0.287, P =0.004), RVD (r = 0.450, P < 0.001), LVEDD (r = 0.248, P = 0.013) and LVEF (r = 0.232, P = 0.020). Multivariate logistic regression revealed that miR-21 significantly influenced LA dilation in elderly patients with EH (P < 0.05). CONCLUSIONS Circulating serum levels of miR-21 are increased in elderly patients with EH with LA dilation. miR-21 levels are significantly correlated with LA dilation in elderly patients with EH, and miR-21 may be a factor related to the clinical pathophysiological occurrence of and treatment for the progression of hypertension-related early heart damage in EH patients.

Hypertension is an important public health concern among the elderly population worldwide, and it is associated with an increased risk of adverse cardiovascular events. As the global population ages, the prevalence of hypertension and damage to target organs,including the heart, eyes, brain, kidneys, and vasculature, continue to increase.[1]Currently, hypertension is the most important risk factor for cardiac diseases, such as coronary atherosclerosis, heart failure, and atrial fibrillation. Left ventricular hypertrophy (LVH) is an initial compensatory mechanism that occurs in response to hypertension-induced cardiac stress; LVH can progress to heart failure and is considered an independent cardiovascular risk factor. However, clinicians have increasingly focused on left atrium (LA) remodelling in hypertension.[2]The LA is highly sensitive to minimal haemodynamic changes in the left ventricle (LV),such overloading vessel pressure induced by hypertension.[3]Alterations in the LA structure and function may precede detectable LV dysfunction; thus,parameters related to the LA could potentially serve as earlier and more accurate biomarkers of disease than parameters related to the LV.[4]Hypertension,ischaemic heart disease, atrial fibrillation (incident and prevalent), incident stroke, and cardiovascular disease-related mortality were associated with a larger LA maximum volume. Measures of LA structure and function, independent of LV metrics, are significantly associated with key prevalent and incident cardiovascular outcomes.[5,6]LA dilation occurs earlier than LVH, and it might serve as a sensitive surrogate endpoint for determining cardiovascular mortality and morbidity rates in patients with essential hypertension (EH).[2]

MicroRNAs (miRNAs) are small noncoding RNAs that function as key posttranscriptional regulators of gene expression in all eukaryotic cells.miRNAs can control physiological and pathological processes that are fundamental to a wide variety of cardiovascular diseases, including hypertension.[7]Currently, several miRNAs have been shown to perform antihypertrophic functions and act as agonists of the hypertrophic response in hypertensive cardiac damage.[8,9]Kontaraki,et al.[10]revealed that miR-21,miR-1, miR-133a, miR-26b, miR-208b, miR-499, and miR-1 have distinct expression profiles in hypertensive patients relative to healthy individuals and are associated with clinical indices of LVH in hypertensive patients. Among these related miRNAs,we found that miR-21 plays an important role in the development of the cardiovascular system and the occurrence of diseases, and it is usually highly expressed in both animal models and the plasma or tissues of humans with hypertension.[11,12]Moreover,miR-21 is associated with fibrogenesis in multiple organs, and inhibition of miR-21 expression prevents hypertrophic stimulation-induced cardiac remodelling.[13,14]

Therefore, in the present study, serum miR-21 levels were compared between groups of elderly patients with EH with or without LA dilation, and the value of miR-21 levels in the early diagnosis of heart damage (LA dilation) in elderly EH patients was further evaluated; these studies provided a basis for the early diagnosis and treatment of hypertensive cardiac remodelling.

METHODS

Ethics Statement

All the experimental procedures were approved by the Ethics Committee of Hebei General Hospital[clinical ethics approval number (2019027)]. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines, as defined by the International Conference on Harmonisation. Written informed consent was obtained from all the patients before the study.A data and safety monitoring committee were established to provide oversight of safety and efficacy considerations. The study was registered on ResMan [approval number ChiCTR1900026699].

Study Population

This cross-sectional study included 100 EH patients[15]who were recruited from Hebei General Hospital between November 2019 and March 2021;this population included 32 males and 68 females who were between 65 and 97 years of age. All the patients provided a detailed medical history and underwent comprehensive clinical examinations and laboratory tests. The exclusion criteria were as follows: acute coronary syndrome, remote myocardial infarction, systemic inflammatory disease, renal failure, autoimmune diseases, cancer, liver diseases,moderate or severe aortic or mitral valvular regurgitation or stenosis, prosthetic valve, atrial septal defect or aneurysm, atrial fibrillation (AF) or conduction disturbances, and inadequate echocardiographic images. Based on their left atrial diameters(LADs), the EH patients were divided into the LA dilation group [LAD >40 (male)/37 (female) mm]and the no-LA dilation group [LAD ≤ 40 (male)/37 (female) mm].[16,17]

Measurement of Circulating miR-21 Levels and Biochemical Assays

We collected 3-4 mL of peripheral blood from each patient into a procoagulation tube (HEBEI XINLE SCI&TECH CO., Ltd, Hebei, China), and the sample was centrifuged at 2000 r/min for 10 min.The supernatant was transferred to a clean EP tube and further centrifuged at 13,000 r/min for 10 min to remove intact chromatin from the ruptured blood cells.The supernatant was transferred to another clean EP tube and stored at -80 °C. MiRNAs were extracted from the prepared blood using a miRNeasy Serum/Plasma Advanced Kit (Qiagen, Germany),strictly following the manufacturer’s instructions.Quantitative RT-PCR was performed as described in the manual (Qiagen, Germany). The primers for miRNA used in this study were purchased from Qiagen. The specimen with the lowest Ct value for the miR-21 gene was used as a control. The results for each serum specimen were calculated with the 2-ΔΔCtmethod. Cel-miR-39 was used as an exogenous control, as previously reported by other studies.[18,19]

Fresh blood samples were analysed using a Beckman AU5800 clinical chemistry autoanalyzer to measure the levels of serum creatinine (Scr), blood urea nitrogen (BUN), uric acid (UA), fasting blood glucose (FBG), total cholesterol (TC), triglyceride(TG), high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C), lipoprotein (a) [Lp(a)], apolipoprotein A1 (apoA1), and apolipoprotein B (apo B). The estimated glomerular filtration rate (eGFR) was calculated with the CKDEPI Creatinine 2009 equation.

Echocardiographic Examination

All the echocardiographic examinations were performed by a clinical board-certified cardiologist,who was blinded to the clinical data of the study population, using a cardiac ultrasound machine (Epiq7C,Philips Medical Systems, Andover, MA, USA). All the patients were in sinus rhythm at the time of examination, and all the measurements were calculated from three consecutive cycles. The averages of the three LAD, interventricular septum (IVS), right atrium diameter (RAD), right ventricle diameter(RVD), left ventricular end-systolic diameter (LVESD), left ventricular end-systolic diameter (LVEDD)and left ventricular ejection fraction (LVEF) measurements were recorded.

Statistical Analysis

Data were analysed using SPSS version 26.0(SPSS Inc.; Chicago, IL, USA.). Before analysis, all the data were evaluated by the Kolmogorov-Smirnov test for distribution normality and then presented as either the mean ± SD for normally distributed continuous variables or the median (quartile range, 25%-75%) for nonnormally distributed continuous variables. Differences in clinical variables with normal distributions between the two groups were analysed with a two-samplettest for continuous variables. The categorical data from the two groups that did not have a normal distribution are expressed as ratios or percentages, and the chisquare test was conducted to compare the groups.Differences in continuous measurement data between the two groups were analysed with the Mann-WhitneyUtest. Associations between miR-21 level/LAD and biochemical levels were analysed by Spearman correlation. Multivariate logistic regression models were used to evaluate the associations of the level of miR-21 with LA dilation in elderly EH patients. The biomarker measurements were logtransformed for the logistic regression analyses.Multicollinearity was examined using the variance inflation factor (VIF) among the included continuous variables in the logistic model. If none of the VIF values are greater than 10, collinearity is not a problem. Calibration of the logistic model was assessed using the Hosmer-Lemeshow test, whereby we considered a value ofP< 0.05 to indicate that the model had poor calibration. In this analysis, we adjusted for sex, Scr level, and LVEF (model 1) and for sex, Scr level, LVEF, RVD, Lp(a) level, cardiac diastolic dysfunction, cerebral infarction, and diabetes mellitus (model 2) based on factors withP< 0.05 andP< 0.20, respectively, to distinguish between the two groups. Finally, a receiver operating characteristic (ROC) curve was generated, and the area under the curve (AUC), sensitivity, specificity, and Youden index were determined to estimate the diagnostic value of miR-21. Statistical significance was defined asP< 0.05.

RESULTS

Patient Demographics

As shown in Table 1, there were no significant differences in the mean age, duration of hypertension, hypertension classification, or proportions of patients with coronary atherosclerotic heart disease,diabetes mellitus, hyperlipidaemia, cerebral infarction, infection, proteinuria, cardiac diastolic dysfunction, or hypothyroidism between the LA dilation and no-LA dilation groups (allP> 0.05). However, there were more male patients (24 of 58) in the no-LA dilation group than in the LA dilation group(8 of 42) (P =0.018). Several patients were bedridden due to multiple morbidities and age. Thus, the body mass index of these patients could not be accurately obtained, so we did not present data about patient body mass index here.

Table 1 Clinical characteristics of individuals in no-LA dilation and LA dilation groups.

Expression of Serum Biomarkers and Clinical Factors in the Patients

Compared with those in the no-LA dilation group, the levels of miR-21 [8.02 (5.21, 14.39)vs.6.05 (3.81, 8.95)P= 0.011] and LVEF (6 7.02±3.82vs.64.14±4.43,P= 0.001) were higher in the LA dilation group, and the levels of Scr [70.40(64.45,80.15)vs. 6 3.9(60.1,73.43)],P= 0.020] were higher in the no-LA dilation group. Conversely, no significant differences between the two groups were found in the levels of TC, TG, HDLC, LDLC, VLDLC, Lp(a),apoA1, apoB, FBG, BUN, UA, eGFR, IVS, RAD,RVD, LVESD or LVEDD (P> 0.05) (Table 2).

Correlations of Serum miR-21 Levels, LAD, Biochemical Indices, and Other Echocardiographic Parameters

Figure 1 shows that the levels of HDLC (r=-0.209,P= 0.037), apoA1 (r= -0.269,P= 0.007) and RAD (r= 0.203,P= 0.043) were significantly correlated with miR-21 expression. The LAD was significantly correlated with the RAD (r= 0.287,P= 0.004),RVD (r= 0.450,P< 0.001), LVEDD (r= 0.248,P=0.013) and LVEF (r=0.232,P= 0.020). However,there were no significant correlations between miR-21 level/LAD and other lipid indices or echocardiographic parameters.

Figure 1 Correlations between miR-21/LAD and other biomarkers. miR-21 levels by real-time PCR correlated significantly with LDL(A), HDLC (B), apoA1 (C) and RAD (D) in 100 elderly essential hypertensive patients. The LAD correlated significantly with RAD(E),RVD(F), LVEDD(G) and LVEF(H) in 100 elderly essential hypertensive patients. The miR-21 variable was log-transformed. LAD: left atrium diameter; LDL: low density lipoprotein; RAD: right atrium diameter; RVD: right ventricle diameter; LVEDD: left ventricular end systolic diameter; LVEF: left ventricular ejection fraction.

Figure 2 ROC analysis of the miR-21 for diagnose the possibility of LA dilation in elderly essential hypertensive patients.AUC: area under the curve; LA: left atrium; ROC: receiver operating characteristic.

Multivariate Analysis with Logistic Regression

Among the variables that could distinguish the two groups (Tables 1 and 2), the miR-21 level was significantly associated with LA dilation in the unadjusted model. Furthermore, model 1 adjusted for sex, Scr level, and LVEF and showed significant differences between the two groups (P< 0.05, Tables 1 and 2). Model 2 adjusted for sex, Scr level, LVEF,RVD, Lp(a) level, history of cardiac diastolic dysfunction, cerebral infarction, and diabetes mellitus,which are thought to contribute to LA dilation (P<0.20, Tables 1 and 2).

In the adjusted models, miR-21 level showed a significant odds ratio in model 1 (OR = 4.916, 95%CI: 1.291-18.713) and model 2 (OR = 5.571, 95% CI:1.311-23.678) (Table 3). The Hosmer-Lemeshow tests showed significant goodness of fit for model 1(P= 0.491) and model 2 (P = 0.283). The C-statistic was 0.761 (95% CI: 0.669-0.853,P< 0.001) and 0.840 (95% CI: 0.763-0.917,P< 0.001) for model 1 and model 2, respectively.

Table 2 Expression of the serum biomarkers between none-LA Dilation and LA dilation groups.

The Value of the miR-21 Level for LA Dilation Diagnosis in Elderly Patients with EH

The effect of the miR-21 level on the diagnosis of LA dilation is presented in Fig 2. The results for the AUC, sensitivity, specificity, and Youden index value are presented. For the miR-21 levels, an AUC of 0.649 (95% CI: 0.539-0.759), a Youden index of 0.240, a sensitivity of 61.90%, and a specificity of 62.07% were obtained.

DISCUSSION

Hypertensive-mediated organ damage is a disease that develops silently and evolves progressively over several years, and it is involved in the development of functional and structural alterations in the arterial bed, the central nervous system, the heart, and the kidneys. In fact, it is possible that at the time of hypertension diagnosis, almost all patients have already suffered from hypertensive target organ damage (TOD).

Currently, several biomarkers related to hypertensive TOD, such as vascular stiffness, coronary artery calcium, and inflammation, have been studied to evaluate their potential use in assessing cardiac damage in hypertensive patients. Previously,many studies have mainly focused on LVH as the TOD in the heart due to hypertension[20][21][22]. LVH is an adaptive response of myocardial cells to chronically increased afterload in the heart and is a strong predictor of heart failure. However, LVH has been shown to not be sensitive enough for the early detection of heart damage.[23]Recently, an increasing number of clinicians have focused on LA remodelling in hypertension target cardiac damage.LA dilation occurs earlier than LVH and has been considered an independent risk factor for cardiac events, including atrial fibrillation and heart failure.[3]

An increasing number of recent studies indicate that miRNAs, such as miR-1, miR-21, miR-133, miR-145, miR-505, and miR-510, are important in the pathogenesis of EH.[7][24][25]In recent years, miR-21,which is one of the most intensively studied miRNAs,is highly expressed in the organs and tissues of mammals and has been indicated to be closely related to EH. Lu,et al.[26]found that miR-21 was highly expressed in the hypertrophic left ventricle and the proximal medullary cortex tissue of fibrotic kidneys in the SHR model. MiR-21 is involved in the pathological process of cardiac hypertrophy and renal fibrosis induced by hypertension.[13]Our recent study also found that miR-21 is present at high levels in chronic heart failure patients with kidney injury.[27]Thus, in this research, we studied the relationship of serum miR-21 levels and LA dilation in elderly patients with EH to determine the possibility of using miR-21 in the early diagnosis of hypertensive heart damage.

Table 3 Multivariate analysis using logistic regression: predictive factors for the risk of LA Dilation in elderly essential hypertension. The miR-21 variable was log-transformed. Model 1: Adjusted for gender, Scr and LVEF (P < 0.05 factors in Table1 and 2); model 2:Adjusted for gender, Scr, LVEF, RVD, Lp(a), the history of cardiac diastolic dysfunction, cerebral infarction, diabetes mellitu (P < 0.20 factors in Table1 and 2). CI: confidence interval; LA: left atrium; Lp(a): lipoprotein (a); LVEF: left ventricular ejection fraction; OR: odds ratio; RVD: right ventricle diameter. Scr: creatinine.

The global population is ageing rapidly. Several epidemiological studies report an association between the ageing population and the development of coexisting morbidities;[28]therefore, elderly individuals exhibit a high prevalence of multimorbidity.[29]The presence of 8 coexisting morbidities(coronary artery disease, diabetes mellitus, hyperlipidaemia infarction, hypothyroidism, infection,proteinuria and cardiac diastolic dysfunction) was examined in our study. The morbidity rate was not significantly different between the no-LA dilation and LA dilation groups (P> 0.05). Although these illnesses might influence miR-21 expression and LAD, there was no significant difference between the two groups, which excluded the influence of these diseases for the models in our study.

Our results showed that the LA dilation group had a significantly lower level of Scr (Table 2), which might be due to early glomerular hypertension and hyperfiltration in EH. Researchers found that hypertension could induce renal haemodynamic stress, activation of signalling pathways and neurohormones, systemic inflammation, diffuse endothelial dysfunction, and oxidative stress.[21][30][31]Brenner,et al.[32]identified glomerular hyperfiltration as a key mediator of progressive kidney damage. At the single-nephron level, hyperfiltration is hypothesized to be an early link in the chain of events that lead from intraglomerular hypertension to albuminuria and, subsequently, to reduced GFR.[33]Furthermore, inhibition of the renin-angiotensin system was found to be effective in preventing glomerular hypertension and hyperfiltration, as well as in reducing albuminuria and kidney damage.Moreover, the low proportion of male patients, who have higher muscle mass than females, decreased the level of Scr in the LA dilation group.

Impaired LV relaxation and elevated LA pressure are common observations in patients with hypertension.[34]High BP-induced early diastolic LV dysfunction can be observed in patients before the development of LV hypertrophy.[35]The LV of the heart fills by two separate mechanisms: (1) earlydiastole: LV fills passively through active relaxation; (2) late-diastole: remaining blood contributing to the total end-diastolic volume enters the ventricle via active contraction of the LA.[36]In this study, we found that the LVEF in the LA dilation group was increased (Table 2), which might be due to the increased compensatory LA volume in the latediastole stage.

In the correlation analysis, a negative correlation between miR-21 and HDL-C/apo A1 was observed.Studies have revealed the role of miRNAs in the regulation of cholesterol metabolism.[37][38]Circulating miR-21-5p levels act as a novel diagnostic biomarker in adult male patients with metabolic syndrome.[39]In the T2DM rat model, miR-21 exacerbated lipid metabolic disorder by increasing the levels of TG, TC, and LDL-C and decreasing the level of HDL-C.[40]Because HDL-C is formed by the interaction of lipid-poor apoA1 with the cellular integral membrane protein adenosine triphosphatebinding cassette transporter ABCA1,[41]apoA1 overexpression decreased atherosclerosis progression in mouse models. Currently, apoA1-reconstituted HDL or apoA1 mimetic peptides have been utilized in multiple preclinical and clinical trials.[42][43][44]

Additionally, we found a significant positive correlation between serum miR-21 levels and RAD in our elderly patients with EH (Figure 1D). Several comorbid conditions, including EH, can directly affect RV function without having an impact on afterload. Chang,et al.[45]showed that circulating miR-21 levels are associated with the severity of RV dysfunction in patients with hypoxia-induced Pulmonary arterial hypertension. In addition, Reddy,[46]et al. noted that miR-21 levels might serve as a plasma biomarker for RV failure under conditions of volume and pressure overload in a murine model of pulmonary insufficiency and stenosis.

However, no significant linear correlation between miR-21 levels and LAD was observed. This may be due to the confounding factors in the model indicated in the logistic regression analysis (Table 3)and the dynamic changes in the miR-21 levels of patients with different stages; this would be consistent with the findings of Chang,et al.,[47]who showed that upregulation of miR-21 in the early phase(RV hypertrophy) and downregulation in the late phase (RV dysfunction) under conditions of pulmonary arterial hypertension triggered a biphasic regulation of cardiac remodelling and cardiomyocyte apoptosis.

Interestingly, our data revealed a positive correlation between RAD/RVD and LAD in elderly patients with EH (Figure 1 E-F). Jessie,et al.[48]explored RV/RA function in preserved ejection fraction(HFpEF) and found that elevated RA pressure and stiffness in HFpEF-pulmonary hypertension were explained by changes in the LA-RA interaction, and increased RA pressure and stiffness are an indication of HFpEF severity. In addition, EH contributes to heart failure with HFpEF, increased RV afterload and pulmonary vascular resistance. These results show the possibility that RA dilation may be an earlier sign of hypertensive cardiac remodelling.

Finally, in our present study, we found that the level of miR-21 was significantly higher in the LA dilation group than in the no-LA dilation group. In the univariate and multivariate logistic regression analysis, we proved the relationship between miR-21 levels and LA dilation in hypertension patients,and miR-21 levels might be a new diagnosis/prognosis indicator of early hypertensive heart damage. However, the AUC of miR-21 in the diagnosis of hypertensive LA dilation was only 0.649 (95% CI:0.539-0.759). However, although the diagnostic efficacy of miR-21 was not as good, a close relationship with early cardiac remodelling in hypertension was indicated in our research. In addition, apoA1 levels, HDL-C levels and RAD were significantly correlated with miR-21 expression, indicating the potential interaction among miR-21, lipid metabolism and right heart remodelling in EH. In future research, we should pay attention to the early role of miR-21 in hypertensive heart damage using nucleotide biologics to slow hypertensive cardiac injury progression.

There are some limitations in our study. This was a single-centre study that included a relatively small number of patients. In addition, there was a lack of serial echocardiograms to assess LAD changes over time in relation to hypertension in these patients. Furthermore, this is a study on Chinese patients with EH, which might limit the generalizability of our findings. Thus, further large-sample prospective multicentre studies may be necessary to prove the diagnostic/prognostic role of miR-21 in early hypertensive heart damage.

In conclusion, circulating serum miR-21 levels are increased in elderly patients with EH with early heart damage. Circulating miR-21 levels have a significant relationship with early heart damage in elderly patients with EH and may be a factor that is related to the clinical pathophysiological occurrence of and treatment for the progression of hypertensive early heart damage.

Acknowledgements

This work was supported by the 2019 Hebei Science and Technology Project (grant number19 277787D); 2019 Hebei Innovation Capability Promotion Project (grant number 199776249D). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no competing interests.