Abstract
Rationale. Cardiac-specific troponins (cTns) are excellent circulating biomarkers and considered the current gold standard for a rapid diagnosis of acute myocardial infarction (MI). Nevertheless, abnormal cTn do not differentiate between the different aetiologies of myocardial injury – including type 2 MI (T2MI). In fact, there is no biomarker allowing to differentiate type 1 MI (T1MI) and T2MI. Cardiac myosin-binding protein C (cMyBP-C) was discovered to be released earlier than cTn upon myocardial injury. Initial results of its clinical application in the detection of MI has suggests a potential benefit towards cTn in sensitivity and specificity. The development of more sensitive protein biomarker assays results in continuous improvements of detectability, extending the range of clinical applications to the detection of subclinical cardiovascular disease (CVD). On the other hand, these efforts have not yet led to improvements in risk assessment compared to existing risk scores. Non-coding RNAs (ncRNAs) are expressed in a tissue- and/or cell-type specific manner and show surprising stability in the circulation and ideal properties of circulating biomarkers. They have been assessed as biomarkers, and microRNAs (miRNAs) have attracted most attention. More recently, other ncRNA classes have been identified, including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) and have been implicated as novel biomarkers in acute settings of MI but their relationship to myocardial protein biomarkers has not been described. Additionally, ncRNAs have been proposed as prognostic biomarkers for cardiovascular disease. ncRNAs are quantified via quantitative real time polymerase chain reaction (RT-qPCR), which is known to be affected by the presence of heparin in the used biomaterial. Their release kinetics in MI have not been explored without confounding by heparin. Besides their exploration as circulating biomarkers for acute MI, ncRNAs have not yet been reported in the differential diagnosis of specific MI subtypes such as T2MI.Objective. To compare ncRNA species in heparinase-treated samples with established and emerging protein biomarkers for myocardial injury. To combine protein and ncRNA biomarkers to identify novel biomarker candidates to further discriminate different MI aetiologies.
Methods and Results. Screening of 158 circRNAs and 21 lncRNAs in human cardiac tissue identified 12 circRNAs and 11 lncRNAs as potential biomarkers with cardiac origin. 11 miRNAs were included. Human myocardial tissue was spiked into plasma from healthy individuals and the expression levels of ncRNAs were determined and compared to cardiac proteins. At low spike-in concentrations of myocardial tissue, significantly higher regression coefficients were observed across ncRNA species compared with cTn and cMyBP-C. We transferred our findings to the clinical setting of transcoronary ablation of septal hypertrophy (TASH), in which the exact time point of myocardial injury is known and biomarker kinetics can be assessed in a highly controlled setting. We assessed protein and ncRNA molecules in early time points after TASH as well as in a MI. Heparinase-treatment of serial plasma and serum samples of patients undergoing TASH removed spurious correlations between miRNAs in non-heparinase-treated samples. After TASH, muscle-enriched miRNAs (miR-1 and miR-133a) showed a steeper and earlier increase than cardiac-enriched miRNAs (miR-499 and miR-208b). Putative cardiac lncRNAs, including previously as a cardiac lncRNA described LIPCAR, did not rise, refuting a predominant cardiac origin. We performed a circRNA microarray screening and assessed candidate circRNAs in MI and myocardial injury samples. Cardiac circRNAs remained largely undetectable. In a validation cohort of acute MI, receiver operating characteristic curve analysis revealed noninferiority of cardiac-specific miRNAs compared with gold standard cTn, but miRNAs failed to identify cases presenting with low cTn values. cMyBP-C was validated as a biomarker with highly sensitive properties, and the combination of muscle-enriched miRNAs with high-sensitivity (hs) cTnT and cMyBP-C returned the highest area under the curve values for the diagnosis of MI. Next, we sought to explore biomarkers for their potential to differentiate different MI subtypes such as T2MI and acute myocardial injury (AI) according to the latest Universal Definition of Myocardial Infarction (UDMI4). While cTn allows for rapid diagnosis of T1MI, its performance to differentiate acute AI or T2MI is limited. The objective was to combine biomarkers to improve discrimination of different MI aetiologies. We determined levels of cTnT and cTnI, cMyBP-C, NT-proBNP and ten miRNAs, known to be associated with cardiac pathology in a total of n=495 serial plasma samples at three time points (on admission, after 1h and 3h) from 57 non-ST-elevation myocardial infarction (NSTEMI), 18 AI, and 31 ST-elevation myocardial infarction (STEMI) patients, as defined by UDMI4 and 59 control individuals. We then applied linear mixed effects model to compare the kinetics of all molecules in these MI subtypes. Established cardiac necrosis markers (cTnT, cTnI) and novel cardiac necrosis markers (cMyBP-C) performed relatively well in discriminating NSTEMI from AI but failed in differentiating T1MI vs T2MI at early time points. All cardiac necrosis markers were higher in T1MI than in T2MI at 3h after admission. Muscle-enriched miRNAs (miR-1 and miR-133a) were correlated with cardiac necrosis protein markers but did not offer better discrimination. Established cardiac stress marker NT-proBNP differentiated AI and T1MI at all time points but failed to discriminate between T1MI and T2MI. However, the combination of NT-proBNP and cTnT along with age presented discriminative potential in differentiating T1MI, T2MI and AI returning an overall AUC of 0.76 [95%CI 0.67 - 0.84].
Conclusions. In a comparative assessment of ncRNAs and protein biomarkers for myocardial injury, cMyBP-C showed properties as the most sensitive cardiac biomarker while miRNAs emerged as promising candidates to integrate ncRNAs with protein biomarkers. Sensitivity of current miRNA detection is inferior to cardiac proteins but a multi-biomarker combination of muscle-enriched miRNAs with cMyBP-C and hs-cTnT could open a new path of integrating complementary characteristics of different biomarker species. Rather than using single biomarkers of myocardial necrosis, a combination of clinical biomarkers for cardiac necrosis (cTn) and cardiac strain (NT-proBNP) might aid in differentiating T1MI, T2MI and AI.
| Date of Award | 1 Apr 2023 |
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| Original language | English |
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| Supervisor | Manuel Mayr (Supervisor) & Anna Zampetaki (Supervisor) |