Novel diagnostic potential of miR-1 in patients with acute heart failure.

BACKGROUND: A number of circulating micro-ribonucleic acids (miRNAs) have been introduced as convincing predictive determinants in a variety of cardiovascular diseases. This study aimed to evaluate some miRNAs' diagnostic and prognostic value in patients with acute heart failure (AHF).
METHOD: Forty-four AHF patients were randomly selected from a tertiary heart center, and 44 healthy participants were included in the control group. Plasma levels of assessed miRNAs, including miR -1, -21, -23, and -423-5-p were measured in both groups. The patients were followed for one year, and several clinical outcomes, including in-hospital mortality, one-year mortality, and the number of readmissions, were recorded.
RESULTS: An overall 88 plasma samples were evaluated. There was no significant difference in terms of demographic characteristics between the AHF and healthy groups. Our findings revealed that mean levels of miR-1, -21, -23, and -423-5-p in AHF patients were significantly higher than in the control group. Although all assessed miRNAs demonstrated high diagnostic potential, the highest sensitivity (77.2%) and specificity (97.7%) is related to miR-1 for the values above 1.22 (p = 0.001, AUC = 0.841; 95%CI, 0.751 to 946). Besides, the levels of miR-21 and -23 were significantly lower in patients with ischemia-induced HF. However, the follow-up data demonstrated no significant association between miRNAs and prognostic outcomes including in-hospital mortality, one-year mortality, and the number of readmissions.
CONCLUSION: The result of our study demonstrated that miR-1, -21, -23, and -423-5-p can be taken into account as diagnostic aids for AHF. Nevertheless, there was no evidence supporting the efficacy of these miRNAs as prognostic factors in our study.

1. Introduction

Acute heart failure (AHF) is one of the most life-threatening and burdensome cardiovascular diseases (CVD) in both developed and developing countries [1, 2]. The prevalence of heart failure in 2020 was over six million patients in the United States of America (U.S.A), which is estimated to rise to more than eight million patients by 2030 [3]. The timely and accurate diagnosis of AHF and distinguishing it from chronic heart failure (CHF) are of great importance [4-6]. However, it is not always feasible to make a definite diagnosis merely based on history, physical examination, and echocardiogram [7]. Therefore, further investigations to establish novel biomarkers are warranted in this regard.

Despite well-established biomarkers, including brain (B-type) natriuretic peptide (BNP) and N-terminal pro-BNP (NT-proBNP) for assigning the diagnosis of AHF, there are some limitations in this era to apply as reliable indicators. For instance, a series of patient characteristics and conditions such as older age, obesity, renal dysfunction, atrial fibrillation, thromboembolic events, etc., can affect serum levels of BNP/ NT-proBNP and may lead to false results [8, 9]. Moreover, a multicenter trial on 1100 high-risk subjects with systolic HF reported no beneficial effect of using NT-proBNP-guided strategy within the routine outpatient management of those patients [10].

Micro-RNAs (miRNAs) are small non-coding RNAs with 21-25-nucleotide to regulate various genes [11]. Since their discovery in 1993 in nematode Caenorhabditis elegans, their role in several physiological and pathological conditions has been widely studied [12-17]. After that, further studies introduce a spread of biological effects of miRNAs within the circulatory system, including their role in cardiac fibrosis and hypertrophy, which are essential HF pathophysiologies [18]. Some miRNAs are closely related to the pathophysiology of HF.

MiR-1 is one of the most abundant miRNAs in cardiac tissue and is linked to cardiac remodeling being inversely correlated with hypertrophy. Experimental animal studies showed that adenoviral delivery of miR-1 was able to reverse hypertrophy [19, 20]. It plays also a role in atrial and ventricular arrhythmias. For example, it has been shown that its levels are markedly reduced in patients with atrial fibrillation. It also plays a role in acute coronary syndromes, a hypothesis that has yet to be further validated [21].

MiR-21 plays a role in inflammation and smooth muscle proliferation and fibrosis. It has been found to be increased in mice hearts subjected to ischemia-reperfusion and more specifically in fibroblasts within the infarct zone [19, 21]. It plays also a profibrotic role in heart failure from causes other than ischemia (i.e. aortic stenosis) through induction of matrix metalloprotease-2 (MMP-2) production [19, 20]. On the other hand, it attenuates cardiomyocyte apoptosis secondary to oxidative stress [20].

MiR-23 is upregulated in cardiac hypertrophy and promotes hypertrophy, while antagomiR-mediated silencing of miR-23a reverses it [20, 21].

Finally, miR-423-5-p has been also found to be increased in HF. In a small study, Tijsen et al. showed that miR-423-5-p strongly discriminated HF-associated dyspnea from healthy controls (C-statistic 0.91) and non-HF dyspnea (C-statistic 0.83) and was correlated with NT-proBNP and LVEF [22]. In another study comparing several miR levels in patients with dilated cardiomyopathy and age- and sex-matched healthy controls, miR-423-5-p was found to have modest discrimination power (C-statistic 0.67) and was also correlated with NT-proBNP levels [23]. A 2020 systematic review showed that miR-423-5-p was among 5 miRNAs that were differentially expressed in more than one included studies [24]. Furthermore, miR-423-5-p levels in plasma may be time-dependent. A study of 246 post-MI patients showed that miR-423-5-p levels rose over time from index hospitalization to 3 months and up to 1 year post-discharge [25].

On the role of miRNAs in HF prognosis, a very recent systematic review and meta-analysis showed that HF patients with low expression of miR-1, miR-21, miR-23, and miR-423-5-p have significantly worse overall survival. However, among them, miR-423-5-p is the stronger biomarker for HF prognosis [26].

Many experimental animal, preclinical, and clinical human studies and reviews have tried to explore the role of miR in HF diagnosis with much attention focused on their relative superiority or non-inferiority compared to established biomarkers such as BNP or NT-proBNP [20, 21]. MicroRNAs may predict the development of HF at an earlier stage as they play a pathophysiological role in myocardial hypertrophy, fibrosis and apoptosis prior to overt clinical symptoms. Despite the positive results of some studies, the clinical utility of these miRNAs in clinical practice is yet to be determined. Here, we aimed to evaluate the potential of selected miRNAs, including miR-1, -21, -23, and -423-5-p, in the diagnosis and prognosis of AHF patients. These miRNAs were mainly selected as the most representative miRNAs that play different roles in cardiovascular pathology: hypertrophy, inflammation, fibrosis, acute coronary syndrome, and arrhythmia.

2. Methods

This study consisted of two phases. In the first phase, the plasma samples of the AHF patients and the healthy control group were evaluated to detect the levels of assessed miRNAs, including miR -1, -21, -23, and -423-5-p. Also, baseline demographic and clinical characteristics associated with the level of miRNAs were recorded. In the second phase, patients were followed for one year after admission, and several outcomes, including in-hospital mortality, one-year mortality, number of readmissions, and Functional Classification of New York Heart Association (FC) after one year, were recorded ().

2.1. Inclusion/Exclusion criteria

Patients who were admitted to a tertiary hospital with a diagnosis of either acute decompensation of CHF (International Classification of Diseases version 10 [ICD-10] code, I50.23: acute on chronic systolic [congestive] heart failure) or de novo AHF according to Framingham-based criteria following the confirmation of two attending cardiologists were included. Patients with HF with reduced ejection fraction (HFrEF: at least one major Framingham-based criteria/two minor criteria and ejection fraction [EF] <40%) and those with HF with preserved ejection fraction (HFpEF: at least one major criteria/two minor criteria and EF ≥50%) were included. As per established HF guidelines, heart failure with mid-range EF (HFmrEF) was defined as HF with an EF of 40–49%, and those patients with HFmrEF were excluded from our study [27]. Patients with HFmrEF were excluded as this phenomenon is considered a gray zone that shares some characteristics with HFpEF patients and some other characteristics with HFrEF patients, and could influence the outcome. Finally, 44 patients were randomly selected from AHF patients, and 44 healthy control were selected from the hospital staff or patients’ companions.

2.2. Plasma sample

Five-milliliter blood samples were obtained within 24 hours after admission by a direct venous puncture into sodium citrate-containing tubes. The samples were stored at -80°C and were subjected to defrost/unfreeze.

2.3. RNA isolation

The process of RNA extraction was conducted using Tripura isolation reagent (Roche Inc., Germany), following the manufacturer’s protocol. Agarose gel electrophoresis was used to assess the integrity of the extracted RNA. Moreover, the extracted RNA was further evaluated by NanoDrop 2000c UV-Vis spectrophotometer (Thermo, MA, USA).

2.4. Deoxyribonucleic acid (DNA) syntheses and real-time polymerase chain reaction (RT-PCR)

Deoxyribonucleic acid (DNA) syntheses and real-time polymerase chain reaction (PCR) for quantifying the relative miRNA levels in blood samples in this study were conducted as previously described [28]. In brief, a miRCURY™ LNA™ miRNA RT Kit (Exiqon Inc, Massachusetts) was utilized for cDNA synthesis using 10 ng of the extracted RNA based on the manufacturer’s instructions. The reactions were conducted at a final volume of 10 μl in a T100 Thermocycler system (Bio-Rad, USA). A light cycler 96 system (Roche Inc., Germany) was utilized to quantify the mature miRNAs and use the ExiLENT SYBR Green master mix (Exiqon Inc, Massachusetts) and miRNAs specific primer sets. PCR was repeated for the second time to control the procedure’s integrity and normalized by U6 expression levels considering the required controls (such as non-template control and no reverse transcription). The thermocycling conditions for quantitative PCR were as follows:

1 cycle of 50°C for 2 min, 95°C for 10 min, 40 cycles of 30 sec at 94°C, and 30 sec at 60°C.

U6 small nuclear RNA was considered as the endogenous control for normalization. The 2-ΔCt method (mentioned below) was used to determine the relative expression levels of target miRNAs.

ΔCt = Ct target gene–Ct U6

2.5. Ethical consideration

This study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki, and the ethics committee approved the protocol. The ethical approval for this study was issued from the Tabriz University of Medical Sciences with the ethics committee number: IR.TBZMED.REC.1397.142. Written informed consent was obtained from all participants after a brief description of the aim of the study.

2.6. Statistical analysis

All categorical data were reported as frequency and percentage and numeric data as mean ± SD (). The level of selected miRNAs is presented as fold-change relative to controls (mean level of miRNAs in the control group assumed to be equal to 1). Student t-test was used to compare two groups in data with normal distribution and the Mann-Whitney test for non-normal data or small sample size. ANOVA or Kruskal-Wallis tests were used to compare more than two groups in data with normal and non-normal distributions or small sample sizes, respectively. Receiver operating characteristic (ROC) curves were generated to estimate the area under the curve, sensitivity, and specificity. For the cutoff point values with the highest Youden index, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated. The association between the miRNAs’ level and patients’ outcome during follow-up was evaluated using multivariable logistic regression analysis by adjusting to the age, sex, smoking, and FC at admission.

The sample size was calculated using the formula of Buderer et al. [29] with an expected sensitivity of 0.95, expected specificity of 0.95, prevalence of 0.20, precision of 0.15, confidence level of 95%, and expected drop out of 15% resulting in a final sample size (with 15% dropout) of 49 patients [30].

3. Results

A total of 88 plasma samples were evaluated, including the plasma samples from 44 AHF patients and 44 healthy participants ().

No significant difference was seen between the two groups in terms of the baseline characteristics, including age, gender, body mass index (BMI), smoking, and place of residence (p>0.05; ). Ischemic heart disease, hypertension, and diabetes were present in 17 (38.6%), 12 (27.2%), and 8 (18.1%) AHF patients, respectively. The mean EF in all AHF patients was 21.75 ± 10.3%. Forty patients (90.9%) had HFrEF, and the majority of patients had the FC of III or IV at presentation (34.1 and 43.2, respectively). Twenty-nine patients (65.9%) had AHF due to ischemia, and de novo AHF was detected in 16 patients (36.4%; ).

The mean relative level of miR-1 in the AHF group was 1.930 ± 0.830 (p<0.00, ). Its sensitivity and specificity for the values above 1.22 were 77.2% and 97.7%, respectively (p = 0.001, AUC = 0.841; 95%CI, 0.751–946; ; ). Relative miR-21 level in AHF patients was on average 1.445 ± 0.534 (P = 0.002) with the sensitivity and specificity for the values above 1.12 of 70.4% and 86.3%, respectively (p = 0.001, AUC = 0.857; 95%CI, 0.783–0.932). The mean relative level of miR-23 AHF was 1.490 ± 0.545 (p = 0.002). Its sensitivity and specificity for the values above 1.24 were 70.4% and 97.7%, respectively (p = 0.001, AUC = 0.837; 95%CI, 0.746–0.928). Relative miR-423-5-p level in AHF patients was on average 1.681 ± 0.546 (P <0.001) with the sensitivity and specificity for the values above 1.39 of 72.7% and 93.1%, respectively (p = 0.001, AUC = 0.881; 95%CI, 0.805–0.957; ; ). Accordingly, miR-1 had the highest net reclassification improvement index (NRI of miR-1, 0.35; 95%CI, 0.25 to 0.46; ).

Regarding the left ventricle function, the plasma levels of miR-21 and miR-423-5-p were higher in patients with HFpEF than in those with HFrEF, although these differences were not statistically significant (p = 0.775, p = 0.760, respectively). Also, the levels of miR-1 and miR-23 were lower in patients with HFpEF than those with HFrEF, which were not statistically significant (p = 0.353, p = 0.420, respectively) (). Also, no significant difference in the levels of studied miRNAs was detected between AHF patients with de novo and acute on CHF (). The miR-21 and miR-23 were significantly lower in patients with ischemia-induced HF when compared to the non-ischemic group (p = 0.027 and p = 0.029, respectively). However, no significant differences were observed in the levels of miR-1 and miR-423-5-p ().

The levels of selected miRNAs in different categories of acute heart failure.

A; The levels of selected miRNAs in two types of heart failure (heart failure with reduced ejection fraction [HFrEF] and with heart failure with preserved ejection fraction (HFpEF]), B; The levels of selected miRNAs with respect to the type of heart failure (de Novo vs acute on chronic), C; The levels of selected miRNAs with respect to the cause of heart failure (ischemia-induced vs non-ischemia induced).

Thirty-five patients were followed up (loss to follow-up = 20%). Of these patients, 3 patients (6.8%) died at their initial hospitalization, and 7 patients deceased during the follow-up (median, 1 month; ranging from 1 to 7 months after discharge). The leading cause of death was the decompensation of HF in all patients. Of the surviving patients (n = 25), 9 patients (25.7%) had not been hospitalized in one year after admission, and 16 patients (45.7%) had been readmission an average of 3 times. Four patients were candidates for heart transplantation, but no cardiac transplant was performed. Patients’ NYHA FC after one year is reported in . The majority of the surviving patients had an FC I (44%), indicating no physical activity limitations. There was no association between the level of miRNAs prognostic outcomes ().

4. Discussion

One of the main advantages of miRNAs, which are proposed as novel biomarkers for the diagnosis of a variety of CVD, is that they are detectable in various body fluids such as blood, saliva, urine, milk, amniotic fluid, etc. [31]. This feature enables us to conveniently detect the changes and track them down to find the etiologies without performing invasive procedures. Despite miRNAs possessing some advantages rather than the well-known biomarkers, e.g., NT-proBNP, they are not applied in the clinical setting. It is worth noting that some risk factors indicated to affect the level of NT-proBNP, including age, obesity, gender, and renal function, have no substantial effect on the dynamic levels of prognostic/diagnostic miRNAs [32]. The current study investigated the clinical significance of the assessed miRNAs, including miR-1, -21, -23, and -423-5-p, in the diagnosis and prognosis of AHF. We found a significant elevation of these miRNAs in the plasma samples of AHF patients compared to the healthy control subjects, which display substantial diagnostic potential and significantly high sensitivity and specificity. The highest sensitivity and specificity were particularly observed in miR-1. Also, miR-23 had a similar specificity to miR-1 but the sensitivity was lower. MiRNAs may predict the development of HF at an earlier stage as they play a pathophysiological role in myocardial hypertrophy, fibrosis and apoptosis prior to overt clinical symptoms. In this way, the main application of miR in HF diagnosis may be in the form of a screening test. When designing a screening test, one needs a high sensitivity (≥ 80%) in order to reduce the false negative rate. Otherwise, many diseased persons will be missed. Thus, the selection of the most appropriate cut-off criterion depends on the purpose of the diagnostic test. Modest sensitivity was detected for miR-1, miR-23, and miR-423-5-p. Nevertheless, all four miRNAs had remarkably high PPV, making them reliable for confirming AHF diagnosis. On the other hand, only the NPV of miR-1 was considerably high to rule out the patients with low probability of AHF with high accuracy.

A few studies, having investigated the diagnostic role of miR-1, -21, -23, and -423-5-p in AHF, yielded some controversial results [22, 33–36]. Unlike our findings, Corsten et al. failed to detect any dynamic changes in the level of miR-1 and -21 in AHF in comparison with healthy controls [32]. In parallel with this, Seronde et al. also postulated that the levels of miR-21 and -23 were similar among patients with AHF, stable CHF, and non-AHF with dyspnea [34]. Moreover, it has been documented that miR-1 was significantly lower among either AHF or stable CHF patients than non-AHF with dyspnea. Zhang et al. also demonstrated a significant diagnostic value of miR-21 for CHF with >99% sensitivity and 97.5% specificity [37]. Furthermore, a recent meta-analysis revealed that miR-423-5-p is an appropriate diagnostic biomarker for HF, with a pooled sensitivity of 81% and a pooled specificity of 67% [38]. Hereto, our findings further proved the diagnostic potential of these miRNAs in AHF patients.

Moreover, we explored the levels of miR-1, -21, -23, and -423-5-p in association with the ischemia-induced HF, left ventricle function, and the history of CHF (de novo or acute on chronic). However, we could detect no significant differences in the levels of assessed miRNAs, except for miR-21 and miR-23, which were significantly lower in patients with ischemia than in those with non-ischemic AHF. in line with our findings, Elis et al. also did not find any significant differences in the levels of miR-23 and miR-423-5-p between patients with HFrEF and HFpEF [33]. Likewise, Seronde et al. demonstrated no significant differences between de novo and acute on chronic AHF patients [34].

Notably, one-year follow-up of AHF patients did not show any efficient prognostic potential of the studied miRNAs regarding in-hospital mortality, one-year mortality, and the number of readmissions. Previously, Seronde et al. also failed to detect any prognostic value for miR-1, -21, -23, and -423-5-p in predicting readmission and one-year mortality [34]. Despite up-regulation of miR-21 during HF, Cakmak et al. also highlighted that it had no potential to be considered a prognostic value [39]; whereas Zhang et al. demonstrated a significant correlation of miR-21 with patients’ two-year mortality but not with readmission rate [10]. Also, it has been reported that an increased level of miR‑21 at the time of clinical compensation (chronic stable compensated state) was directly associated with better two-year survival and longer re-hospitalization‑free status [40]. Noteworthy, higher miR‑423‑5p between the time of admission and clinical compensation was associated with fewer hospital readmissions in two years. Consequently, the time of acquisition of plasma samples can trigger a new development in the prognostic utility of miRNAs.

4.1. Study limitations

Some limitations are imposed on this study due to the challenges of detection and measurement of miRNAs. There are difficulties in measuring absolute levels of miRNAs (as opposed to relative expression vs controls), lack of standardized protocols, intra- and inter-laboratory variation, different types of samples (race, plasma, serum, and tissue), disease stage, etc. The exact time of sampling is crucial but it is yet to be standardized: i.e. miR-423-5-p may increase over time during follow-up. Also, the presence of arrhythmias (such as AF) may alter the levels of miR-1. Finally, in the case of acute coronary syndromes patients are usually on heparin; but heparin has been shown to be a significant inhibitor of PCR-based reactions (such as miRNA analysis) [41].

Since most of the patients had HFrEF in this study, the participation of the patients was completely random and not based on the HF type. In addition, the relatively small sample size in our study was another restriction in accurately evaluating the level of miRNAs among AHF patients with divergent characteristics. Also, we had a partly high rate of loss to follow-up (20%), reflecting presumable bias in the analysis of the patient’s follow-up data. Indeed, 20 percent is suggested to be an acceptable rate for loss to follow-up [42]. On the other hand, limited financial support did not allow us to include more patients. Thus, events per variable ratios for some of our prognostic outcomes (particularly in-hospital mortality) were extremely low and the interpretations should be performed with caution and future studies with larger sample sizes are warranted to admit our findings for the prognostic value of these miRNAs.

Finally, it is suggested to compare AHF with other patients with dyspnea as well as healthy control groups in future studies.

5. Conclusion

In summary, our data demonstrated a remarkable diagnostic power of selected miRNAs (i.e., miR-1, -21, -23, 423-5-p) for AHF. Therefore, these miRNAs in particular miR-1 can be taken into account as diagnostic aids for AHF. Moreover, our results do not support the prognostic value of these miRNAs in AHF. Nevertheless, future large-scale studies are warranted to further elaborate on the prognostic value of these miRNAs. NT-proBNP is still the most convincing biomarker for AHF; therefore, our study might be considered a cornerstone for pioneering more investigations to establish the application of miRNAs as biomarkers for AHF detection in the clinic.

Flowchart of inclusion of patients and follow up protocol.

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Flowchart of statistical analysis plan and approach.

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The performance comparison and validation of models for each miRNA for diagnosis of acute heart failure.

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The level of miRNAs in patients who developed/not developed the adverse events during follow up.

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1 Jul 2022

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Please clarify what the authors mean for ICD Implementation. Is it Implantation or upgrade? Also clarify the timing of ICD implantation and, when correcting, using it as a time dependent variable or removing it from the outcomes.

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Reviewer #1: May I congratulate the authors on conducting this original review. The reviewed manuscript fits with the Journal's scope, with a moderate clinical importance in the cardiology area and adds value to its field. In the review article entitled "Novel Diagnostic Potential of miR-1 in Patients with Acute Heart Failure", the authors try to assess the prognostic and diagnostic role of some miRNA in acute heart failure patients. The article summarises the risk factors and comorbidities of HFpEF and HFrEF patients and compares the plasma values for miR -1, -21, -23, and -423-5-p in acute heart failure patients and healthy subjects. The patients were followed for one year, including in-hospital mortality, one-year mortality, and the number of readmissions and the measures performed seem to have a diagnostic value.

The title is informative and relevant and the research/review question is clearly outlined, however, the authors should consider introducing HFmrEF in the title and in the text as well.

The background is clear and tries to shape what is already known about this topic, however it should offer a clearer view why did the authors only assessed this particular types of miRNA: -1, -21, -23, and -423-5-p for acute heart failure patients.

The main section needs an appropriate reporting: the data could be presented in a better way (data from the tables and figures could be more clearly presented).

Titles, columns and rows are labelled correctly and clearly, however, categories could be grouped more appropriately. The text is repetitive of the data included in tables, although the authors do not convince the reader what is a statistically significant result.

The conclusions section answers the aims of the study although it should be better supported by results. The manuscript should state its impact on current and future practice and also its limitations (eg: what is this assay influenced by, etc).

The references used are relevant, not so recent, referenced correctly and the authors tried to include appropriate key studies, although missed some.

Overall, the review was appropriate to answer the aim, based also on previous studies, however, the authors should address the following issues/ flaws of this article:

1. All acronyms and abbreviations must be explained in full at first mention (e.g DNA, ). All abbreviations used must be defined and spelt out in full in the caption.

2. Author(s) should be consistent when reporting abbreviation throughout the manuscript (eg: HfrEF (line 82) vs HFrEF (line 172,174); or HfpEF (line 83) vs HFpEF (line 172, 174, 186, 236, 254)

3. In the section "prevalence and incidence of HFpEF", in paragraph 3, starting with line 50, the reader could get confused as the authors start comparing HFpEF with HFrEF

4. In the section entitled "Risk factors and comorbidities of HFpEF", the authors should try and restate the ESC guidelines/ first sentence could be reworded.

5. Although the review should refer to HFpEF the authors compare HFpEF with HFrEF (line 50, 72, 96, 104, 113,135, 158, 165, 171, 230.232, 251,253,279,338, 339)

6. Authors should clarify why patients with HFmrEF= heart failure with mildly reduced ejection fraction where excluded from the study – line 84-85 (also that is not a sentence).

7. In study limitation, authors mention that most of the patients included in their study had HFpEF, however, as per the result section, most of them had HFrEF.

8. Authors should state the limitation more clearly (e.g.what influences the assay/ the test?)

Language:

1. "cronray" should read as "coronary" (line 63)?

2. “freeze-thawing” should read as “defrost/unfreeze”? (line 90)

3. A sentence should end with dot “.”(line 90)

4. A sentence always starts with capital letter. Authors should reconsider/ re-arrange first sentence in Discussions section. (line208). Also line 235.

5. Authors should reconsider second sentence in the conclusion section, line 266: “Therefore, they the miRNAs in particular miR-1 can be taken into account as diagnostic aids for AHF.”

Tables and figures:

1. For tables and figures, all the abbreviation used should be defined in a footnote (e.g. FC)

2. Any Table (Table 2) that require more than 2 pages, must be supplied as supplementary material.

3. When reporting in table 1 BMI that should be reported as 23.55 ±6.14

4. In the healthy control group all the subjects were non-smokers?

5. In table 1, when referred to Type of heart failure (based on history), n (%) - “acute on chronic” should read as” acute or chronic”?

6. Table 2 needs a better discussion in the text.

7. Authors should consider performing a statistical analysis plan and should consider using cluster approach.

Reference:

1. The first reference used is the guidelines, however it is advisable to use an updated version (current one dates from 2021)

2. The references used could be updated and more recent.

Reviewer #2: The authors submitted a research article in which they evaluated the potential of selected miRNAs, including miR-1, -21, -23, and -423-5-p, in the diagnosis and prognosis of AHF patients. They randomly selected 85 AHF patients and 58 healthy control individuals. The authors evaluated a signature of miRNAs (miR-1, miR-21, miR-423-5-p and miR-23) and did not find any association between the level of miRNAs prognostic outcomes, whereas diagnostic power of evaluated

miRNAs (i.e., miR-1, -21, -23, 423-5-p) for acute HF were determed. Although these findings are impressive, I would like to put forwrd several issues to comments.

1. Methodology: the authors should clearly report what HF was evaluated: chronic HF after recovery of acute HF or acute de novo HF / acutely decompensated HF. Endpoints are needed to be explained. Please, check and add more information. Flow chart with clear inclusion / non-inclusion criteria a re needed.

2. Statistics: sample size is required to be calculated. Please, add pertnent formula and an example of estimation. Yet, the validation method is needed to be reported along with comparissions of the models with AUCs, IDI and INR.

3. Results: Table 1 does not corresponds to the initial hypothesis of acute HF. The authors should re-check the data and improve them.

4. Please, use comparisson of the model with standard model that is required to be used in accordance to current ESC guideline

5. The multivariate log regression is needed to evaluate whether additional variables exist and influence prognosis.

**********

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Reviewer #1: No

Reviewer #2: No

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13 Aug 2022

Dr. Emily Chenette

Editor-in-Chief

PLOS ONE

Dear Dr. Chenette,

Subject: Revised Manuscript, Authors' Responses

Thank you for your email and favorable response and comments. We appreciate the reviewers’ constructive suggestions. Please find attached the revised manuscript.

Major revisions have been made to improve the manuscript, as suggested.

The revisions and new citations have improved the content. Reformatting and editing also made a seamless flow of information.

The changes are marked by Track Changes.

Below are the authors’ responses to the comments of the reviewers.

We hope that these revisions are acceptable and the manuscript can go forward for publication in PLOS ONE.

Thanks and Regards,

Reza Badalzadeh, PhD

Cardiovascular Research center

Madani Hospital, Tabriz University of Medical Sciences

Tabriz, Iran

Tel: +984133373919

Fax: +984133373910

Email: reza.badalzadeh@gmail.com

Authors' Responses are colored in blue

Editor Comments:

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Specifically, check the statistics performed and adjust with proper methods. Report the event-per variable ration and the strategy to avoid overfitting. Please report the comparison for diagnostic accuracy in the ROC curves.

The event-per-variable ratios for those with multivariable binary regression analysis were added to the footnote of Table 3. A statement regarding this issue was added to the limitation of the study section. Although it is believed that the evidence underlying the rule of EPV = 10 as a minimal sample size criterion for binary logistic regression analysis is weak [1].

Comparison for diagnostic accuracy in the ROC curves of each miRNA was added as a new column to Table 2.

Comment:

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The manuscript format was changed to meet the style of PLOS ONE

Comment:

2. Please amend your current ethics statement to address the following concerns:

a) Did participants provide their written or verbal informed consent to participate in this study?

b) If consent was verbal, please explain i) why written consent was not obtained, ii) how you documented participant consent, and iii) whether the ethics committees/IRB approved this consent procedure.

“Written informed consent was obtained from all participants.” This statement was added to the ethics statement section.

Comment:

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Regarding the “Data Availability statement”, we added a reasonable phrase fit into this query, in the end of the manuscript before references, if it is not acceptable we can also prepare the minimal data set as supplementary files, if not, the required data could be uploaded in a standard repository system for public access. (https://data.4tu.nl/info/en/use/publish-cite/upload-your-data-in-our-data-repository)

Comment:

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

The ORCID iD was added.

Comment:

5. Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure.

Figure 1 was referred to in the results section, paragraph 3.

Additional Editor Comments:

Please clarify what the authors mean for ICD Implementation. Is it Implantation or upgrade? Also clarify the timing of ICD implantation and, when correcting, using it as a time dependent variable or removing it from the outcomes.

The ICD implementation was removed from the outcomes.

Comment:

Please report the HFA-PEFF and the HF2PEF scores before classifying HFpEF.

Heart failure with preserved ejection fraction was defined based on left ventricular ejection fraction in this study. Considering that E/e′ ratio was not measured for our patients we are unable to report HFA-PEFF and the HF2PEF scores.

REVIEWER 1

Comment:

May I congratulate the authors on conducting this original review. The reviewed manuscript fits with the Journal's scope, with a moderate clinical importance in the cardiology area and adds value to its field. In the review article entitled "Novel Diagnostic Potential of miR-1 in Patients with Acute Heart Failure", the authors try to assess the prognostic and diagnostic role of some miRNA in acute heart failure patients. The article summarises the risk factors and comorbidities of HFpEF and HFrEF patients and compares the plasma values for miR -1, -21, -23, and -423-5-p in acute heart failure patients and healthy subjects. The patients were followed for one year, including in-hospital mortality, one-year mortality, and the number of readmissions and the measures performed seem to have a diagnostic value.

The title is informative and relevant and the research/review question is clearly outlined, however, the authors should consider introducing HFmrEF in the title and in the text as well.

We highly appreciate your comments and positive feedback.

HFmrEF definition was added to the methods section and related reference was cited.

Comment:

The background is clear and tries to shape what is already known about this topic, however it should offer a clearer view why did the authors only assessed this particular types of miRNA: -1, -21, -23, and -423-5-p for acute heart failure patients.

A thorough explanation of the reasons for the selection of these miRNAs was added to the introduction section. These miRNAs were mainly selected to include the most representative miRNAs that play different roles in cardiovascular pathology: hypertrophy, inflammation, fibrosis, acute coronary syndrome, arrhythmia:

MiR-1 is one of the most abundant miRNAs in cardiac tissue and is linked to cardiac remodeling being inversely correlated with hypertrophy. Experimental animal studies showed that adenoviral delivery of miR-1 was able to reverse hypertrophy [2, 3]. It plays also a role in atrial and ventricular arrhythmias. For example, it has been shown that its levels are markedly reduced in patients with atrial fibrillation. It also plays a role in acute coronary syndromes, a hypothesis that has yet to be further validated [4].

MiR-21 plays a role in inflammation and smooth muscle proliferation and fibrosis. It has been found to be increased in mice hearts subjected to ischemia-reperfusion and more specifically in fibroblasts within the infarct zone [2, 4]. It plays also a profibrotic role in heart failure from causes other than ischemia (i.e. aortic stenosis) through induction of matrix metalloprotease-2 (MMP-2) production[2, 3]. On the other hand, it attenuates cardiomyocyte apoptosis secondary to oxidative stress [3].

MiR-23 is upregulated in cardiac hypertrophy and promotes hypertrophy, while antagomiR-mediated silencing of miR-23a reverses it [3, 4].

Finally, miR-423-5-p has been also found to be increased in HF. In a small study, Tijsen et al. showed that miR-423-5-p strongly discriminated HF-associated dyspnea from healthy controls (C-statistic 0.91) and non-HF dyspnea (C-statistic 0.83) and was correlated with NT-proBNP and LVEF [16]. In another study comparing several miR levels in patients with dilated cardiomyopathy and age- and sex-matched healthy controls, miR-423-5-p was found to have modest discrimination power (C-statistic 0.67) and was also correlated with NT-proBNP levels [5]. A 2020 systematic review showed that miR-423-5-p was among 5 miRNAs that were differentially expressed in more than one included studies [6]. Furthermore, miR-423-5-p levels in plasma may be time-dependent. A study of 246 post-MI patients showed that miR-423-5-p levels rose over time from index hospitalization to 3 months and up to 1 year post-discharge [7].

On the role of miRNAs in HF prognosis, a very recent systematic review and meta-analysis showed that HF patients with low expression of miR-1, miR-21, miR-23, and miR-423-5-p have significantly worse overall survival. However, among them, miR-423-5-p is the stronger biomarker for HF prognosis [8].

Many experimental animal, preclinical, and clinical human studies and reviews have tried to explore the role of miR in HF diagnosis with much attention focused on their relative superiority or non-inferiority compared to established biomarkers such as BNP or NT-proBNP [3, 4]. MicroRNAs may predict the development of HF at an earlier stage as they play a pathophysiological role in myocardial hypertrophy, fibrosis and apoptosis prior to overt clinical symptoms. Despite the positive results of some studies, the clinical utility of these miRNAs in clinical practice is yet to be determined. Here, we aimed to evaluate the potential of selected miRNAs, including miR-1, -21, -23, and -423-5-p, in the diagnosis and prognosis of AHF patients.

Comment:

The main section needs an appropriate reporting: the data could be presented in a better way (data from the tables and figures could be more clearly presented).

The design of the tables was improved to make them more clear. Nevertheless, upon you find it inadequate or have any specific suggestions please kindly guide us with more detailed explanation.

Comment:

Titles, columns and rows are labelled correctly and clearly, however, categories could be grouped more appropriately. The text is repetitive of the data included in tables, although the authors do not convince the reader what is a statistically significant result.

The data in tables were grouped to show the results more clearly. The results section was improved accordingly.

Comment:

The conclusions section answers the aims of the study although it should be better supported by results. The manuscript should state its impact on current and future practice and also its limitations (eg: what is this assay influenced by, etc).

The conclusion was edited and the impact of the findings of our study was added. The limitations of measurement and detection of miRNAs as added to the study limitation section.

Comment:

The references used are relevant, not so recent, referenced correctly and the authors tried to include appropriate key studies, although missed some.

The references were updated accordingly.

Comment:

Overall, the review was appropriate to answer the aim, based also on previous studies, however, the authors should address the following issues/ flaws of this article:

1. All acronyms and abbreviations must be explained in full at first mention (e.g DNA, ). All abbreviations used must be defined and spelt out in full in the caption.

The abbreviations and acronyms were defined in the text and also the caption of Tabels.

Comment:

2. Author(s) should be consistent when reporting abbreviation throughout the manuscript (eg: HfrEF (line 82) vs HFrEF (line 172,174); or HfpEF (line 83) vs HFpEF (line 172, 174, 186, 236, 254)

The consistency of abbreviation was checked and the mentioned mistakes were corredcted..

Comment:

3. In the section "prevalence and incidence of HFpEF", in paragraph 3, starting with line 50, the reader could get confused as the authors start comparing HFpEF with HFrEF

We could not find this section in our manuscript. Would you please check again the section? It may have been already corrected or it could be related to another manuscript.

Comment:

4. In the section entitled "Risk factors and comorbidities of HFpEF", the authors should try and restate the ESC guidelines/ first sentence could be reworded.

We could not find this section in our manuscript. Would you please check again the section? It may be related to another manuscript.

Comment:

5. Although the review should refer to HFpEF the authors compare HFpEF with HFrEF (line 50, 72, 96, 104, 113,135, 158, 165, 171, 230.232, 251,253,279,338, 339)

We could not find the mentioned issue in the listed lines in our manuscript. Would you please check again the section?

Comment:

6. Authors should clarify why patients with HFmrEF= heart failure with mildly reduced ejection fraction where excluded from the study – line 84-85 (also that is not a sentence).

An explanation was added to the methods section.

Comment:

7. In study limitation, authors mention that most of the patients included in their study had HFpEF, however, as per the result section, most of them had HFrEF.

The sentence was edited.

Comment:

8. Authors should state the limitation more clearly (e.g.what influences the assay/ the test?)

The limitations section was completed.

Comment:

Language:

1. "cronray" should read as "coronary" (line 63)?

2. “freeze-thawing” should read as “defrost/unfreeze”? (line 90)

3. A sentence should end with dot “.”(line 90)

4. A sentence always starts with capital letter. Authors should reconsider/ re-arrange first sentence in Discussions section. (line208). Also line 235.

5. Authors should reconsider second sentence in the conclusion section, line 266: “Therefore, they the miRNAs in particular miR-1 can be taken into account as diagnostic aids for AHF.”

The language mistakes were corrected.

Comment:

Tables and figures:

1. For tables and figures, all the abbreviation used should be defined in a footnote (e.g. FC)

The abbreviations were accordingly completed.

Comment:

2. Any Table (Table 2) that require more than 2 pages, must be supplied as supplementary material.

Tables were edited to fit on one page. Thank you.

Comment:

3. When reporting in table 1 BMI that should be reported as 23.55 ±6.14

The style of reporting, “the mean ± SD” was corrected.

Comment:

3. In the healthy control group all the subjects were non-smokers?

Thank you for nice query.

The prevalence of smoking among healthy subjects was added.

Comment:

4. In table 1, when referred to Type of heart failure (based on history), n (%) - “acute on chronic” should read as” acute or chronic”?

“Acute on chronic heart failure” was named according to the ICD-10-CM Code: I50. 23 as an acute heart failure occured following the chronic heart failure”.

Comment:

5. Table 2 needs a better discussion in the text.

The discussion over Table 2 was added to the discussion section.

Comment:

6. Authors should consider performing a statistical analysis plan and should consider using cluster approach.

A flowchart of the statistical analysis plan and approach was added as supplementary figure 2.

Comment:

Reference:

1. The first reference used is the guidelines, however it is advisable to use an updated version (current one dates from 2021)

2. The references used could be updated and more recent.

The reference list was updated.

REVIEWER 2

The authors submitted a research article in which they evaluated the potential of selected miRNAs, including miR-1, -21, -23, and -423-5-p, in the diagnosis and prognosis of AHF patients. They randomly selected 85 AHF patients and 58 healthy control individuals. The authors evaluated a signature of miRNAs (miR-1, miR-21, miR-423-5-p and miR-23) and did not find any association between the level of miRNAs prognostic outcomes, whereas diagnostic power of evaluated miRNAs (i.e., miR-1, -21, -23, 423-5-p) for acute HF were determed. Although these findings are impressive, I would like to put forwrd several issues to comments.

Comment:

1. Methodology: the authors should clearly report what HF was evaluated: chronic HF after recovery of acute HF or acute de novo HF / acutely decompensated HF. Endpoints are needed to be explained. Please, check and add more information. Flow chart with clear inclusion / non-inclusion criteria are needed.

We included patients with acute decompensation of CHF (International Classification of Diseases version 10 [ICD-10] code, I50.23: Acute on chronic systolic (congestive) heart failure) or de novo acute heart failure. This explanation was added to the methods section.

Comment:

2. Statistics: sample size is required to be calculated. Please, add pertnent formula and an example of estimation. Yet, the validation method is needed to be reported along with comparissions of the models with AUCs, IDI and INR.

The sample size calculation method was added. Validation models including AUC and INR were also added to the supplementary table.

Comment:

3. Results: Table 1 does not corresponds to the initial hypothesis of acute HF. The authors should re-check the data and improve them.

The data in Table was re-checked and corrected.

Comment:

4. Please, use comparisson of the model with standard model that is required to be used in accordance to current ESC guideline

The AUC for each miRNA is reported for comparison with standard model.

Comment:

5. The multivariate log regression is needed to evaluate whether additional variables exist and influence prognosis.

The multivariate log regression has been already performed but due to the low number of outcomes and the proability of overfitting, we were not able to add more variables to our model. This issue was adderessed in our study limitations section.

References

1. van Smeden M, de Groot JAH, Moons KGM, Collins GS, Altman DG, Eijkemans MJC, et al. No rationale for 1 variable per 10 events criterion for binary logistic regression analysis. BMC Med Res Methodol. 2016;16(1):163. doi: 10.1186/s12874-016-0267-3.

2. Melman YF, Shah R, Das S. MicroRNAs in heart failure: is the picture becoming less miRky? Circ Heart Fail. 2014;7(1):203-14.

3. Wang H, Cai J. The role of microRNAs in heart failure. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2017;1863(8):2019-30. doi: https://doi.org/10.1016/j.bbadis.2016.11.034.

4. Romaine SPR, Tomaszewski M, Condorelli G, Samani NJ. MicroRNAs in cardiovascular disease: an introduction for clinicians. Heart. 2015;101(12):921-8.

5. Fan K-L, Zhang H-F, Shen J, Zhang Q, Li X-L. Circulating microRNAs levels in Chinese heart failure patients caused by dilated cardiomyopathy. Indian Heart J. 2013;65(1):12-6.

6. Peterlin A, Počivavšek K, Petrovič D, Peterlin B. The role of microRNAs in heart failure: a systematic review. Frontiers in Cardiovascular Medicine. 2020;7:161.

7. Bauters C, Kumarswamy R, Holzmann A, Bretthauer J, Anker SD, Pinet F, et al. Circulating miR-133a and miR-423-5p fail as biomarkers for left ventricular remodeling after myocardial infarction. Int J Cardiol. 2013;168(3):1837-40.

8. Yang J, Yang X-S, Fan S-W, Zhao X-Y, Li C, Zhao Z-Y, et al. Prognostic value of microRNAs in heart failure: A meta-analysis. Medicine. 2021;100(46).

Submitted filename: response to reviewers 5.docx

Click here for additional data file.

9 Sep 2022

Novel Diagnostic Potential of miR-1 in Patients with Acute Heart Failure

PONE-D-22-15097R1

Dear Dr. Badalzadeh,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

amended manuscript is acceptable

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #3: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All my questions and comments have been satisfactorily answered. I have no further questions and comments.

Reviewer #3: The authors provided a new version of their article. All comments have been addressed. Now the article is ready for publication.

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Reviewer #1: No

Reviewer #3: No

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15 Sep 2022

PONE-D-22-15097R1

Novel diagnostic potential of miR-1 in patients with acute heart failure

Dear Dr. Badalzadeh:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

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Academic Editor

PLOS ONE

Table 1

Baseline characteristics of groups of acute heart failure patients and healthy control.

		AHF group (n = 44)	Healthy control (n = 44)	P value
Age mean ± SD		55 (20)	48 (15)	0.106
Sex n (%)	Male	33 (75.0)	34 (77.3)	0.803
	Female	11 (25.0)	10 (22.7)
Place of residence n (%)	Urban	31 (70.5)	35 (79.5)	0.325
	Rural	13 (29.5)	9 (20.5)
Body mass index mean ± SD		23.55 (6.14)	26.1 (4.38)	0.060
Smoking n (%)		12 (27.3)	8 (18.6)	0.337
Medical History n (%)	Hypertension	17 (38.6)	-
	Diabetes	12 (27.2)	-
	Ischemic heart disease	8 (18.1)	-
	COPD	3 (6.8)	-
	Congenital myopathy	2 (4.5)	-
	Acute rheumatic fever	2 (4.5)	-
	CRF	2 (4.5)	-
	CVA	2 (4.5)	-
NYHA functional class at admission n (%)	I	0 (0)	-
	II	10 (22.7)	-
	III	15 (34.1)	-
	IV	19 (43.2)	-
Type of heart failure (based on EF) n (%)	Reduced EF	40 (90.9)	-
	Preserved EF	4 (9.1)	-
Type of heart failure (based on history) n (%)	Acute on chronic	28 (63.6)	-
	de novo	16 (36.4)	-
Cause of heart failure n (%)	Ischemic	29 (65.9)	-
	Non-ischemic	15 (34.1)	-

AHF, Acute heart failure; EF, Ejection fraction, COPD, Chronic obstructive pulmonary disease CRF, Chronic renal failure; CVA, cerebrovascular accident; NYHA, New York Heart Association

Table 2

Overview of the diagnostic power of selected miRNAs for acute heart failure.

miRNA	Optimal cut-point value*	Group (n)		AUC (95% CI)	Sensitivity (95% CI)	Specificity (95% CI)	PPV (95% CI)	NPV (95% CI)
		AHF	Healthy control
miR-1	≥ 1.22	34	1	0.841 (0.75 to 94)	77.2 (62.1 to 88.5)	97.7 (87.9 to 99.9)	97.14 (82.9 to 99.5)	81.1 (71.3 to 88.1)
	< 1.22	10	43
miR-21	≥1.12	31	6	0.857 (0.78 to 0.93)	70.4 (54.8 to 83.2)	86.3 (72.6 to 94.8)	83.7 (70.5 to 91.7)	74.5 (64.6 to 82.4)
	< 1.12	13	38
miR-23	≥1.24	31	1	0.837 (0.74 to 0.92).	70.4 (54.8 to 83.2)	97.7 (87.9 to 99.9)	96.8 (81.5 to 99.5)	76.7 (67.6 to 83.9)
	< 1.24	13	43
miR-423	≥ 1.35	32	3	0.881 (0.80 to 0.95)	72.7 (57.2 to 85.0)	93.1 (81.3to 98.5)	91.4 (77.9 to 96.9)	77.3 (67.6 to 84.7)
	< 1.35	12	41

* Relative miRNA level. miR, microRNA; AHF, Acute heart failure; PPV, Positive predictive value; NPV, Negative predictive value; AUC, Area under the curve

Table 3

The association between the level of miRNAs and the patients’ outcomes in follow up.

miRNA		In hospital mortality	Readmission in one year	Mortality in one year	FC after one year
miR 1	OR (95%CI)	0.92 (0.11 to 7.69)	0.99 (0.24 to 3.97)	0.95 (0.36 to 2.54)	0.07 (0.00 to 2.60)
	P value	0.939	0.992	0.931	0.152
miR 21	OR (95%CI)	0.17 (0.00 to 8.62)	1.25 (0.11 to 13.69)	1.50 (0.24 to 9.24)	0.06 (0.00 to 14.27)
	P value	0.383	0.853	0.659	0.321
miR 23	OR (95%CI)	2.02 (0.06 to 68.04)	1.03 (0.15 to 6.88)	1.27 (0.27 to 5.97)	0.33 (0.00 to 13.95)
	P value	0.693	0.970	0.756	0.335
miR 423-5-p	OR (95%CI)	4.70 (0.14 to 150.74)	0.02 (0.00 to 1.40)	1.00 0.(95 to 1.04)	0.10 (0.00 to 15.62)
	P value	0.381	0.072	0.983	0.337

*Multivariate regression analysis by adjusting to age, sex, and FC at the first presentation. Abbreviation: FC, Functional Classification of New York Heart Association (NYHA); miR, microRNA; Events per variable ratios were 0.75, 3.2, and 2.5 for in-hospital mortality, readmission in one year, and mortality in one year, respectively.