miRNA miRNA on the Wall, Who Is the Most Cardio-Specific of Them All? Circulating microRNAs as Potential Biomarkers in Acute Coronary Syndrome

The severe and acute manifestation of coronary artery disease (CAD) is acute coronary syndrome (ACS); therefore, prompt diagnosis can save lives. Cardiac biomarkers that are accepted to use in evaluating ACS are creatine kinase muscle/brain subtype (CK-MB), cardiac troponin I (CTnI), or cardiac troponin T (CTnT). However, these markers have several drawbacks, such as prolonged time to rise for prompt diagnosis and elevation in patients with chronic kidney diseases (CKD). Lately, potential, novel candidates for cardiac ischemia biomarkers have been developed, one of which is micro-ribonucleic acids (miRNAs). miRNAs are potential due to their remarkable reproducibility and stability. Several miRNAs, such as, miR-1, miR-133a/b, miR-208a/b, and miR-499a, greatly rise in concentration in the plasma or serum of patients with acute cardiac ischemia, signifying their cardiac specificity and promising biomarkers in patients with ACS. This systematic review aims to elucidate the role of cardio-specific miRNA in acute myocardial ischemia (AMI) and its relationship with other cardiac biomarkers.


INTRODUCTION
Coronary artery disease (CAD), also recognized as ischemic heart disease (IHD), is the most common cardiovascular disease [1]. Acute coronary syndrome (ACS) is elicited by a complete or partial thrombosis of an artery following the rupture of an atherosclerotic plaque and; ACS attributes to an array of clinical presentations encompassing unstable angina, acute myocardial infarction (AMI), and either ST-segment elevation MI (STEMI) or non-ST-segment elevation MI (NSTEMI) [2]. In general, creatine kinase brain/muscle subtype (CK-MB), cardiac troponin I (CTnI), or cardiac troponin T (CTnT) are employed as biomarkers to assess and diagnose AMI prognosis. However, these markers have several drawbacks. CTnI or CTnT starts to increase in three to four hours, followed by CK-MB that increases in four to six hours following the onset of myocardial injury. These biomarkers are also elevated in patients with chronic kidney diseases (CKD) and can be deceiving even when myocardial ischemia is not clinically suspected [3]. Therefore, a clinical demand for a new biomarker, which possesses the ability to rule out or rule in AMI promptly, is increasing. Micro-ribonucleic acids (miRNAs) appear to be a likely candidate for the prompt diagnosis of AMI. miRNAs are short (~22 nucleotides) endogenous RNAs. miRNAs consisted of about 22 nucleotides, more or less, endogenous RNAs. Thousands of miRNAs have been identified in humans, and they are taught to control one-third of humans' genes. Multiple of them are have been involved in prevalent human pathological conditions [4]. The exceptional stability of miRNAs in urine and blood made them appealing candidates as biomarkers for numerous diseases. Several miRNAs, such as, miR-1, miR-133a/b, miR-208a/b, and miR-499a, were disclosed as greatly elevated in the plasma or serum of patients with AMI [5]. This review aims to elucidate the role of cardio-specific miRNAs in ACS and their relationship with conventional cardiac biomarkers. It also assesses and appraises the capacity of miRNAs as valuable diagnostic biomarkers for prompt diagnosis of AMI. containing valvular heart disease or congenital heart disease are being excluded. Only casecontrol studies are included in this article, as many as 20 in number. Additionally, references of all publications from the initial search are also included for supplementary sources.
After a careful investigation, as many as 20 clinical studies were covered in this review. In total, 3560 subjects were included. The source of extraction of microRNA was plasma in 13 studies. The source of microRNA extraction was plasma in six studies, but only one study (Wang et al., 2011) used both plasma and whole blood as the source of microRNA extraction [6]. reported that compared to patients with unstable angina, patients with STEMI or NSTEMI were found to manifest with greater levels of miR-1, miR133a, and miR-208b [13]. miR-1, miR-133a, miR-133b, and miR-208b were found to be exclusively connected with CTnT levels in a multiple linear regression analysis which incorporated CTnT and clinical variables (p <0.001).
In 2011, Gidlof et al. published that all miRNA levels were considerably more eminent in diseased populations than in those who are normal (p < 0.001) [15]. Although other miRNAs did not associate with EF or CTnT, miR-208b was negatively correlated with ejection fraction (EF) and positively correlated with cTnT [15]. Samples of blood were retrieved at 24 hours, 48 hours, and 72 hours. Within 12 hours of the onset of the symptoms in STEMI patients, the levels of miR-1, miR-133a, miR-208b, and miR-499-5p were increased. Li YQ et al. in 2013 revealed that miR-1, -133a, -208b, and -499 levels were considerably elevated in samples of blood or plasma that are retrieved within 12 hours of the onset of AMI. However, for the diagnosis of AMI, the four results of increased miRNAs levels were not remarkable to CTnT (p ˃0.05) [21].
The other three studies that investigated miR-499 individually found the biomarker to be upregulated [8,22,23]. In 2010, Adachi et al. observed that miR-499 was associated with CK-MB and meaningfully elevated in the AMI group compared to the other groups (p <0.0001).
The blood sampling time was not beyond 48 hours after the onset of chest pain as a symptom [8]. The plasma concentration of miR-499 peaked at between six to 12 hours [8].
Chen et al. in 2015 published a study in which he took blood samples at 0 hours, 12 hours, 24 hours, three days, and seven days after the onset of chest pain [22]. The average duration of the onset of symptoms, which is chest pain, and the emergency room arrival was found to be 4.46±3.36 hours. Compared to those in unstable angina (UA) group and healthy control group, the relative plasma miR-499 level was meaningfully elevated in 53 patients with AMI (2.75±1.39 in UA group, 0.50±0.35 in the healthy control group, and 5.12±2.29 in AMI group).
stated that in MI group, miRNA-499 was meaningfully elevated compared to controls (p <0.05) [23]. Three hours following the onset of chest pain in AMI, mRNA-499 could be identified in the serum, peaked after 12 hours, and progressively decreased after 15 hours [23].
In two studies that examined miR-1, it was found that the biomarker is up-regulated [11,12].
In 2010, Ai et al. described that the level of miR-1 was remarkably elevated and positively associated with the cardiac troponin [11]. Cheng et al. in 2010 elaborated that the level of miR-1 was higher compared to healthy controls (p <0.05) and positively correlated with the level of CK-MB (r=0.68; p <0.05). The blood sampling average time was 8.5±3.82 hours [12].
In 2010, D'Alessandra et al. noticed the upregulation of miR-1, miR-133a, miR-133b, and miR-499-5p, and the downregulation of miR-122 and miR-375 [10]. The levels of miRNAs were remarkably altered as seen in the AMI group compared to the control group (p <0.01).
miRNAs were also found to be correlated positively with CTnI (p <0.01). The average blood sampling time was 517+309 minutes following the onset of AMI. The plasma levels of miR-1, miR-133a, and miR-133b were formerly peaked at T0, or at a time point adjacent to the peak of CTnI. Reversely, miR-499-5p displayed a more gradual time progression and peaked behind the CTnI. In conclusion, the study summarized that after a three-day time development, the levels of miR-1, miR-133a, and miR-133b had recovered back to control levels.

DISCUSSION
Lately, miRNAs are given immense importance in managing autophagy, necrosis, and apoptosis of cardiomyocytes, hence its function in myocardial infarction [26]. Investigations have additionally examined the pieces of evidence that miRNAs are seeped into the circulation from the heart following a myocardial injury, causing a dynamic increase in their levels [27,28]. Consequently, miRNAs which circulate in the plasma or blood have newly surfaced as promising biomarkers for the AMI diagnosis and/or AMI prognosis because of their reproducibility, specificity, and stability.
Our study reviews 20 articles which examine miRNAs in ACS patients.  [7]. In terms of diagnosing AMI, miR-208a possessed greater specificity and sensitivity. miR-208a was readily identified in patients with AMI within four hours since the onset of chest pain. However, compared to CTnI, its diagnostic significance was inferior. No remarkable distinction in sex and age was mentioned in the study. Another study by Adachi et al. in 2010 revealed that plasma concentrations of miR-499 were increased in AMI patients within 48 hours [8]. The concentration was noted to peak in about six hours to 12 hours and later undetectable upon hospital discharge [8]. The upregulation of miR-499 was associated with the level of CK-MB.
Corsten et al. in 2010 found a notable rise in miR-208b and miR-499, correlating positively with the level of CTnI and CTnT [9]. The plasma levels of microRNAs were not influenced by a broad array of clinical confounding factors, which involve age, sex, systolic blood pressure, kidney function, body mass index, and white blood cell count. Another study by D'Alessandra et al. in 2010 presented an increased level of miR-1, miR-133a, miR-133b, and miR-499-5p and a decreased level miR-122 and miR-375, which are also positively correlated with CTnI [10]. It is unusual in a sense that the reduction in the concentration of miR-122 and miR-375 has not been examined in any medical condition, not in humans or the animal models of human diseases.
In 2010, Ai et al. revealed a remarkable elevation in miR-1 in patients with AMI, which also positively correlated with cardiac troponins [11]. An elevation in circulating miR-1 was not correlated with gender, age, blood pressure, diabetes mellitus state, and AMI biomarkers.
Additionally, miR-1 has positively correlated also with the levels of CK-MB [12]. A remarkable elevation of miR-133a and miR-328 concentrations in patients with AMI and the correlation of CTnI with miR-133 or miR-328 concentrations were also confirmed by Wang et al. [6]. Nevertheless, the levels of miR-133 and miR-328 peaked faster compared to CTnI. No statistical deviations were reported between the control groups and the AMI groups for each of the analyzed variables, except for the levels of low-density lipoprotein (LDL) and total cholesterol (TC), which seemed to increase in AMI patients.
The upregulation of miR-1, miR-133a/b, miR-208a/b, and miR-499 levels and their correlation with CTnT was observed by Widera et al. [13]. miR-133a and miR-208b were correlated with all-cause mortality in six months, despite adjusting for sex and age. However, these miRNAs missed their correlation with the end results upon adjustment for hs-CTnT, which showed that, to a sensitive myonecrosis marker, these biomarkers do not contribute to prognostic information.
In 2011, Kuwabara et al. presented the upregulation of miR-1 and miR-133a and their positive correlation with the level of CTnT [14]. Levels of serum miR-1 and miR-133a were found to elevate in Takotsubo [20]. A multivariate analysis was done, and the statistical significance level for all associations was not influenced despite adjustment for sex, age, and sampling time.
The levels of miRNA-1, miRNA-133a, miRNA-208b, and miRNA-499 were found to be elevated significantly in post-AMI patients compared to normal volunteers who were paired for sex and age [21]. Even though positive correlations were observed between cTnT and the four circulating miRNAs in 12 hours after the onset of the symptoms, for prompt diagnosis of AMI, no miRNAs was reported to be superior to CTnT.
The level of serum miR-1 rose swiftly hours after symptoms of AMI [12]. Over 20-fold increase in the level of serum miR-1 was detected within 24 hours of AMI. Furthermore, there was a positive correlation which links CK-MB and serum miR-1. The report also proposed that miR-1 may additionally be associated with the size of myocardial infarction in humans. Chen et al. have also pointed out that plasma levels of miR-499 markedly elevated 12 hours after the onset in patients with AMI, and imposed positive correlation with cardiac biomarkers [22].
Remarkably, they noticed that miR-499 concentration in two-and three-vessel CAD was considerably greater than single-vessel CAD; therefore, the biomarker corresponds positively with coronary stenosis severity. miR-499 levels in patients with AMI in 24 hours following an emergency percutaneous intervention (PCI) were notably more profound than the non-PCI group and those at admission.
Zhao et al. reported that miR-499 concentration in patients with AMI was markedly more elevated than the control group, despite having moderate specificity and sensitivity than CTnI [23]. The half-life of plasma miR-499 is low; its levels markedly elevate in three hours following the AMI onset, peaked at 12 hours, before progressively decreasing. We anticipate that this conclusion would be instrumental for re-infarction diagnosis following an initial AMI.
No notable variation in gender and age within the groups.
As explained by Bialek et al., miR-208a, exclusively synthesized in the heart, rises in conditions such as STEMI and/or myocardial injury induced by reperfusion [24]. During admission (less than three hours of the onset) in STEMI patients when CTnI was not increased yet, miR-208a concentration in plasma elevated to 10-fold increase. miRNA-208a also peaked in advance of both CK-MB and CTnI mass. This condition exhibits a favorable relationship with the traditional biomarkers of myocardial injury.
Lastly, Agiannitopoulos et al. reported a study of Greek AMI patients where the upregulation of miR-208b and miR-499 occurred [25]. An immediate collection of blood samples were performed once the patient is being admitted. Interestingly, the results of the Greek AMI group are concomitant with those from Asian groups, implying that the genetic background does not influence the expression levels of miRNA-208b and miRNA-499. No remarkable distinctions were observed between both groups, particularly in gender, age, smoking condition, and any additional clinicopathological characteristics.

LIMITATIONS
Multiple limitations developed during the development of this study. First, not every study cohort was gender-and age-matched or matched with additional cofounding factors. Second, the population size of some trials was relatively modest. Third, the timing for retrieving blood samples was not discussed in several studies, despite having significance to faithfully label microRNA as a useful cardiac biomarker in the prompt diagnosis of AMI. Fourth, microRNA levels should be investigated in CKD patients to eliminate false positive outcomes. There is a need to carry extensive randomized cohort studies to approach these limitations and assess the potential advantages of having microRNAs as a novel cardiac biomarker.

CONCLUSIONS
Cardio-specific microRNAs (miR-1, miR-133, miR-208, and miR-499) are potent novel biomarkers to perform timely diagnosis and prognosis of acute myocardial infarction. Six serum miRNAs (miR-1, miR-134, miR-186, miR-208, miR-223 and miR-499) posed a greater sensitivity for the diagnosis of angina pectoris. The cardio-specificity of these microRNAs correlates strongly with the conventional cardiac biomarkers and the time when their concentration levels in the blood elevated. Studies involving miR-208b and miR-499 concluded that ethnic and genetic background does not affect the increasing level of miRNAs, the level of both biomarkers correspond positively with the severity of coronary artery stenosis, and that miRNAs may render data on the prognosis of the disease. In acute myocardial infarction cases, the levels of miRNAs rise ahead of the conventional cardiac biomarkers. The reduction of particular miRNAs (miR-122 and miR-375) was detected only in acute myocardial infarction and has not been found in other medical conditions-neither in humans nor animal models. Therefore, additional investigations at greater measure are required to assess the promising role of having microRNA as the novel cardiac biomarker and portray their performance in enhancing the diagnostic strategy in patients with acute coronary syndrome.

DISCLOSURE Funding
None.