miR‐1, miR‐499 and miR‐208 are sensitive markers to diagnose sudden death due to early acute myocardial infarction

Abstract MicroRNAs (miRNAs) are strongly up‐regulated under pathological stress and in a wide range of diseases. In recent years, miRNAs are under investigation for their potential use as biomarkers in cardiovascular diseases. We investigate whether specific cardio‐miRNAs are overexpressed in heart samples from subjects deceased for acute myocardial infarction (AMI) or sudden cardiac death (SCD), and whether miRNA could help differentiate between them. Forty four cases of death due to cardiovascular disease were selected, respectively, 19 cases categorized as AMI and 25 as SCD. Eighteen cases of traumatic death without pathological cardiac involvement were selected as control. Immunohistochemical investigation was performed for CD15, IL‐15, Cx43, MCP‐1, tryptase, troponin C and troponin I. Reverse transcription and quantitative real‐time PCR were performed for miR‐1, miR‐133, miR‐208 and miR‐499. In AMI group, stronger immunoreaction for the CD15, IL‐15 and MCP‐1 antibodies was detectable compared with SCD and control. Cx43 showed a negative reaction with respect to the other groups. Real‐time PCR results showed a down‐regulation of all miRNAs in the AMI group compared with SCD and control. The selected miRNAs presented high accuracy in discriminating SCD from AMI (miR‐1 and miR‐499) and AMI from control (miR‐208) representing a potential aid for both clinicians and pathologists for differential diagnosis.

systematically reviewed by Kong et al 4 who found a great number of studies giving different definitions of SCD grounded, alternately, on time constraints, geographical location of the event, and, finally, on the aetiology of the event SCD. Among others, SCD may be defined as unexpected, non-traumatic death occurring within one hour of the onset of new or worsening symptoms (witnessed arrest) or, if unwitnessed, within 24 hours of last being seen alive. 5 Further difficulties in exactly quantifying the incidence of SCD derive from the fact that most of the available estimates are based on retrospective death certificate-based methodology 4 : Death certificates, vital statistics and census data can misclassify and overestimate SCD. 6 Finally, a further confounding factor is the fact that SCD can be challenging to recognize during the autopsy. Pathologists often refer to sudden unexplained death when an exhaustive post-mortem examination fails to determine a conclusive cause of death. 7 Many SCD cases still remain unexplained even when conventional autopsy and macroscopic and histological investigations are thoroughly performed; consequently, the search for and identification of the cause of death still remain a heavy challenge for pathologists. In recent years, structured autopsy procedures 8  Furthermore, genetic investigations have notably increased diagnostic accuracy in SCD cases 11,12 since genome-wide association studies have demonstrated numerous variations within DNA sequence leading to increased risk of cardiovascular deaths (CVDs). 13 However, a certain quote of CVD genetic risk remains unknown ("missing heritability") and the hypothesis that epigenetic mechanisms could be partially responsible for the "remaining" genetic risk of CVD is increasingly strengthened. 14 Consequently, for both clinicians and pathologists, the search for reliable markers of SCD still remains challenging. 15 In this context, microRNAs (miRNAs) generated enormous enthusiasm for their potential use as biomarkers of several cardiovascular diseases. 16,17 MiRNAs are a class of small non-coding RNAs of ~22 nt that suppress gene expression by hybridizing to the 3' untranslated region of messenger RNA (mRNA), promoting mRNA degradation or disrupting translation, thus acting as post-transcriptional regulators, repressing, or completely silencing, protein translation. Several miR-NAs are strongly up-regulated during pathological stress, and they appear to be aberrantly expressed in blood plasma or serum during the course of many diseases. 18 Among the plethora of miRNAs progressively annotated (about 1500-2000 human miRNAs have been identified), 19 some miRNAs have been demonstrated to play a significant role in cardiogenesis, heart function and pathology, 20,21 thus contributing to the progression of cardiovascular diseases such as cardiac hypertrophy and fibrosis and myocardial infarction. 22,23 Cardiac miRNAs such as miR-1, miR-133a, miR-208a/b and miR-499 are abundantly expressed in the myocardium and are present, stable and detectable in the circulation in different cardiovascular events, [15][16][17][18][19][20][21][22][23][24] such as early after myocardial infarction. 25 MiRNAs have also been explored for cardiovascular risk stratification, 26 thus assuming an increasingly important role as potential cardiovascular biomarkers. 27 Given that specific miRNAs are actively secreted by cardiomyocytes and that cardiomyocyte-derived miRNAs can be found in circulation in various cardiovascular acute events such as AMI and SCD, in the present study, we investigated whether specific cardio-miRNAs are overexpressed in cardiac tissue samples of subjects deceased for AMI or SCD and whether these different cardiac diseases could be differentiated by the miRNA expressed in cardiac tissue samples taken at autopsy.

| MATERIAL S AND ME THODS
The clinical data and autopsy records of the cardiovascular death (CVD) autopsies performed at the Department of Anatomical,

Histological, Forensic and Orthopaedic Sciences-Sapienza
University of Rome and the University of Pisa (Italy) over the period 2013-2017 were evaluated. We selected 44 cases of subjects who died for CVD. We selected only cases with a well-defined clinical course (clinical symptoms, ECG and laboratory data) and with post-mortem confirmed CVD diagnosis. Based on the available clinical and laboratory data, and on the autoptic, histopathological, and immunohistochemical investigation, 19 subjects were classified as having an AMI. AMI diagnosis was determined by a cardiologist based on clinical history, physical examination, electrocardiography and cardiac markers. In all cases, a 12-lead ECG was performed within 10 minutes of arrival into the emergency department. If the standard leads were inconclusive and the patient had signs or symptoms suggestive of ongoing myocardial ischaemia, additional leads were recorded since left circumflex artery occlusion or right ventricular MI could be detected only in V7-V9 and V3R and V4R, respectively. In patients presenting with cardiac arrest or haemodynamic instability of presumed cardiovascular origin, echocardiography was performed immediately following a 12-lead ECG. Cardiac troponin (cTn) and other biomarkers (CKMB) were measured on first assessment and, depending on the patient's survival, repeated 3-6 hours later. An elevated high sensitivity cTn value (>99th percentile URL) was considered diagnostic for AMI. If a cTn assay was not available, an increased CKMB value above the 99th percentile URL was designated as the decision level for the diagnosis of MI. 29 Twenty five subjects who had deceased suddenly, unexpectedly and with no pathogenic findings after thorough macro-and histopathological post-mortem investigations were categorized as SCD.
Survival time ranged from 4 to 6 hours to no more than 12 hours from the abrupt onset of typical symptoms. Control group consisted of 18 cases of traumatic death (survival limited to 12 hours) without cardiac alterations (Table 1). In all cases, toxicology screening was negative for alcohol and drugs of abuse. In all cases, the myocardial samples (standard seven specimens and additional samples taken from macroscopically altered areas) were re-examined histologically to confirm the diagnosis of the cause of death and total RNA, including miRNAs, was extracted from these formalin-fixed paraffin-embedded (FFPE) tissue blocks.

| Reverse transcription and quantitative realtime PCR
Reverse transcription and quantitative real-time PCR were performed for the following miRNAs: miR-1, miR-133, miR-208 and miR-499.

| Statistical analysis
Data were expressed as the mean ± SD from at least three in-

| Immunohistochemistry
Differences in the myocardial structure were found (

| miRNA
Real-time PCR results ( Figure 3) showed a down-regulation of all miRNAs investigated in the AMI group compared with both SCD and control group. Furthermore, significant differences in the expression of miR-1 were found between SCD and controls, being up-regulated in SCD. Notably, miRNAs under analysis were not able to discriminate cases of SCD from subjects deceased for other causes, with the exception of miR-1 that was higher in SCD cases compared to the other two groups.
In order to estimate the diagnostic accuracy of the selected miR-NAs, ROC curve analysis was performed (Figure 4).
Selected miRNAs obtained the best result in discriminating the SCD from AMI cases reaching the highest AUC values (Table 4).
Nonetheless, good results were obtained comparing AMI vs C.

| D ISCUSS I ON
The present study aimed at evaluating the potential diagnostic power of selected miRNAs as tool to differentiate and to quickly orient diagnosis of SCD or AMI. Furthermore, we were interested in proposing miRNAs as candidate markers to improve current diagnosis of SCD or AMI, which is currently performed with the antibody assays present in our work. These antibodies are not the target of selected miRNAs; instead, they represent the current way to discriminate two pathological scenarios. To our knowledge, this study is the first report describing the expression of cardio-miRNAs comparing AMI to SCD cases ( Figure 5). It differs from other investigations that focused on the dysregulation of specific cardio-miRNAs in AMI. 36 We investigated the expression of specific cardio-miRNAs in cardiac tissue samples in subjects deceased for AMI or SCD and whether these different cardiac diseases could be differentiated by the miRNA expression in cardiac tissue samples taken at autopsy.
Our results showed that MCP-1 was strongly expressed in the AMI cases. IL-15 positivity in AMI cases may be interpreted as an expression of the synergism with neutrophilic granulocytes (CD15), and our study confirms the potential for striking cytokine synergy in promoting the local neutrophil response in damaged tissues. [30][31][32][33] The remodelling. 45 Many studies have been performed to characterize those miR-NAs involved in AMI. 36 Yang and colleagues demonstrated that upregulation of miR-1 occurs in structurally diseased human hearts, providing evidence for the role of miR-1 in electrical remodelling and arrhythmias. 38 Furthermore, it has been demonstrated that the inhibition of miR-1 after AMI is able to diminish sudden death in a rat model of surgical myocardial infarction. 46 The expression of these cardio-miRNAs has been often evaluated in bodily fluids from subjects who experienced AMI. For example, in a recent study, miRNAs were measured in plasma from subjects who died in the field, died in hospital or survived to discharge. In that study, plasmatic miR-499-5p was higher in cases with respect to controls. miR-499-5p was lower in both subjects died in hospital and those survived to discharge compared to subjects died in the field. Furthermore, miR-133, miR-208 and miR-499 were lower in subjects survived to discharge compared to those who deceased in the field. 47,48 In several studies, plasmatic concentrations of miR-499 were increased in AMI compared to individuals without cardiovascular diseases. 36  Similarly, serum miR-1 has been reported to significantly increase in patients with AMI compared to normal controls 48 and its level was dropped to normal on discharge following medication. 38 In another study using a rat model, plasmatic miR-208 increased significantly after isoproterenol-induced myocardial injury. 49 Similarly to miR-499, circulating miR-1 has also been shown to increase in blood samples from patients experiencing AMI 38 and a decrease in the infarcted myocardium of a mouse model of AMI was reported. miR-1 has been proposed as an independent biomarker for the diagnosis of AMI and the associated ischaemic arrhythmias. 38 In accordance with our results, a recent quantitative analysis assessing microRNA stability in post-mortem FFPE tissues from subjects dead for AMI compared to healthy controls showed a significant decrease (2.1-fold) in the expression of miR-499a. 50

F I G U R E 5 Experimental outline and results
This fact, together with the increase in miR-499 in serum from patients experiencing AMI, 51,52 supports the hypothesis of its release from the cardiac tissue. Furthermore, in vivo models of AMI demonstrated a decrease in miR-499 levels in the cardiac infarcted zone. 53 In the same study, a decrease in miR-1 in AMI tissues compared to controls was found, although not statistically significant. 50 Contrary to our findings, another study on autoptic infarcted heart tissue from patients with AMI reported an up-regulation of miR-208 in these patients compared to healthy adult hearts. However, a down-regulation of miR-1 and miR-133a in AMI patients compared to healthy adult hearts was reported in accordance with our results.
In the same study, these expression patterns found in myocardial infarction cases were found to be similar to those from foetal hearts, reinforcing the proposed mechanism of cardiac gene reprogramming in the remodelling of the heart. 54 Besides their role in cardiac development and in several cardiac pathological processes, such as cardiac hypertrophy, heart failure, 55-58 cardiomyopathy 59 and angiogenesis, 60  Experimental studies showed that muscle-specific miRNAs as miR-1 and miR-133 may influence the spatial patterns of tissue distribution of ion channels. 64 It has been demonstrated that miR-133 regulates pacemaker channel HCN2 and HCN4 and contributes to the re-expression of these channels in hypertrophic heart. 65,66 In the infarcted area, a significant cytokine up-regulation is detectable due to different pathways like reactive oxygen species generation, complement activation and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation potently stimulate cytokine mRNA (messenger ribonucleic acid) synthesis in both resident and blood-derived cells. 67,68 In infarcted myocardium, an up-regulation of miR-1 that down-regulates the KCNJ2 and GJA1 expressions with the cooperation of miR-206 is present. The first gene encodes the principle pore-forming subunit Kir2.1 of the inwardly rectified ion current (IK1), while the second is responsible for connexin 43, a constituent gap junction protein, fundamental for the impulse propagation and electrical synchronization between myocytes. 68 Our contribution focuses on the diagnostic capacity of miR-NAs in discriminating AMI cases from SCD cases, proposing operational difficulties since SCDs are an area that requires more consistent and more robust studies about miRNAs. Actually, the expression of specific cardio-miRNAs in cardiac tissue samples in subjects deceased for AMI or SCD could be differentiated by the miRNA expression in cardiac tissue samples taken at autopsy. 69 The study of cardio-miRNAs expression may improve clinicians and pathologists' understanding of the mechanisms underlying to cardiac insults and provide targets for future therapies. As has been pointed out, "Those who are working in this field would be well advised to… focus on opportunities where there is a lower barrier of entry for miRNA-based diagnostics, and then perform robust and reproducible studies using biologically applicable samples. Only then will miRNAs succeed as a class of biomarkers for cardiovascular disease". 50 Further studies should be carried out with this specific research purpose.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R S CO NTR I B UTI O N
All the authors listed contributed to the research study design and performed data collection and analysis and interpretation of data. All authors drafted the paper or revised it critically, and they approved the submitted and final versions.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.