Over the last decade, the diagnostic process of DCM etiologies has focused on searching for new biomarkers. An efficient biomarker for DCM should be robust, stable, non-invasive, sensitive, specific to this entity, predictive of a particular DCM etiology, and show a preclinical and clinical relevance to be validated in animal and/or human cell models [19]. We propose the use of peripheral circRNAs as a novel discriminant biomarker of DCM etiologies.
Unlike linear RNA, single circulating circRNAs or circRNAs combined with various other biomarkers are a promising tool for clinical diagnosis of heart diseases, which would improve outcome [20]. Thus, circRNA MICRA was reported to risk-stratify patients after acute myocardial infarction [21]. Peripheral circ_0124644 and circ_0098964 levels have been described as a diagnostic biomarker of coronary artery disease [22]. Related to cardiomyopathies, a set of circulating circRNAs DNAJC6, TMEM56 and MBOAT2 has been proposed to discriminate between healthy and hypertrophic cardiomyopathy [23]. In this sense, hsa_circ_0071542 was upregulated in children with fulminant myocarditis in leukocytes isolated from peripheral blood [24]. Nevertheless, this area remains mostly unexplored in DCM [22, 25]. Hence, further analysis of circRNAs among DCM etiologies might provide early, precise characterization of the disease and lead to novel pathological information, beyond the traditional biomarkers. To the best of our knowledge, the present study is the first to describe a subset of circulating circRNA for a discriminative etiology-based diagnostic in DCM. Circulating hsa_circ_0003258, hsa_circ_0051238 and hsa_circ_0051239 expression levels were upregulated in LMNA-related DCM patients. Notably, hsa_circ_0051238 plasmatic levels were significantly present in the LMNAPh− cohort. Hence, it may be a promising diagnostic biomarker for the early identification of patients in an initial stage of LMNA-related DCM. This will allow personalized therapeutic measures to be applied that help to improve the progression and outcome of LMNA-related DCM. Furthermore, plasmatic hsa_circ_0089762 may provide discriminative power for the ischemic DCM cohort with high-yield diagnostic accuracy and an AUC of 0.92. These circRNAs have been identified mostly in various types of oncologic processes [26–31]. Thus, only hsa_circ_0051239 levels have been upregulated in the myocardium of congenital ventricular septal defect [31]. However, they have not been previously described in DCM cases.
In the current study, circRNA were related to clinical and echocardiographic variables. Male gender, rare non-missense variants in LMNA, and LVEF < 50% have been established as independent factors associated with a more aggressive outcome and even death during follow-up [32]. Herein, all three circRNAs associated with LMNA-DCM etiology were related to male gender [33]. On the other side, echocardiography variables and related circRNAs might suggest a time-evolving sequence. TDI echocardiography is a non-invasive, very sensitive method to assess the cardiac hemodynamic in DCM [34]. TDI reveals that subtle impairment in diastolic myocardial tissue velocities are markers of early cardiac disease and have been associated with outcome in various cardiopathies [16, 17]. In the LMNAPh− group, the E´s TDI is negatively related to hsa_circ_0003258 and hsa_circ_0051239. This E´s TDI impairment suggests an underlying early diastolic dysfunction [35]. A´s TDI in the LMNAPh+ group showed a positive correlation, which indicates that the left atrium is a prominent factor to maintain the LV filling pressure when diastolic dysfunction advances. This sequential TDI septal impairment mirrors the transition from LMNAPh− to LMNAPh+ and may be related to the progressive fibrosis of the interventricular septum located in the basal portion, which is characteristic of the LMNA related-DCM that has been associated with ventricular arrhythmias and worse prognosis [36]. LVEF was independently negatively associated with hsa_circ_0003258, hsa_circ_0051238 and hsa_circ_0051239. According to the LV systolic impairment, hsa_circ_0003258 and hsa_circ_0051238 were related to LV mitral annular plane systolic excursion. Thus, changes in contractility quantified by LV mitral annular plane systolic excursion occur as compensatory mechanisms before impairment of ventricular function [37]. Hsa_circ_0051238 and hsa_circ_0051239 were also negatively related to LVOT velocity, which suggests progressive impairment of the cardiac pump in the LMNAPh+ cohort. Dysfunction of RV is a final common step in DCM and heart failure [38]. RV pressure overload due to PHT, the interventricular interdependence affected by septal fibrosis and underlying ischemia may influence this situation. In support of our results, circRNA, hsa_circ_0003258 was positively increased with the RV lower tricuspid annular plane systolic excursion and PHT [39].
Otherwise, hsa_circ_0089762 correlated to diastolic blood pressure and LVEF in the ischemic group, which supports our results as a specific, highly sensitive biomarker with high-yield diagnostic accuracy. Moreover, hsa_circ_0089762 was related to A´s TDI, which suggests more advanced progression of this entity. Its association with an increase in RV dimension could add information for tailored management in this group, since RV impairment is a worse outcome marker in the ischemic population [40]. In addition, RV involvement has a multifactorial origin that may be influenced by LV remodelling, increased LV filling pressures, the appearance of PHT or RV ischemia [41].
Regarding biological implications, circRNAs spring from introns or exons of their parental genes by back-spliced circularization [25]. Hence, the ratio between linear and circular fractions affects gene expression. According to the protein atlas (proteinatlas.org), parental genes are expressed in cardiac tissue, which supports correlations between etiologies and circRNAs. Hsa_circ_0003258 is synthesized from ZNF652 gene. ZNF652 interacts with CBFA2T3, which acts as a transcriptional repressor [42]. ZNF652 is associated with systolic or diastolic blood pressure and hypertension. However, its role remains unclear [43]. Hsa_circ_0051238 and hsa_circ_0051239 come from the ATP5SL gene. ATP5SL is required for the assembly of mitochondrial NADH: ubiquinone oxidoreductase complex (complex I). Complex I is essential to provide the energy for cardiac function and is related to DCM progression [44]. ATP5SL has been associated with a congenital ventricular septal defect by the overexpression of hsa_circ_0051239 [45]. Finally, hsa_circ_0089762 is generated from the MT-CO2 gene. MT-CO2 is part of the electron transport chain of the mitochondria. Reduced activity of the electron transport chain subunits has been described independently of etiology in ischemic or idiopathic DCM patients [46].
The functional enrichment of the intersecting set of RBSs reveals the role of FMRP in regulation of the membrane potential. Bao et al. described FMRP isoform 1, in rats, as an essential protection factor and a novel potential biomarker in the cardiovascular system[47]. The participation of circRNAs in regulatory networks involving competing-endogenous RNA interactions by sequestering miRNAs has been characterized recently in cardiovascular pathologies [48, 49]. From the set of miRNAs that could be sponged by the circRNAs that we considered, we found significant enrichment in the regulation of focal adhesion and actin cytoskeleton. Both have an important role in human DCM [50], which suggests new pathways of study.
Our current study has several limitations. Firstly, our sample was prospectively recruited from the outpatient clinic. The size of the study sample, comprised of strictly DCM patients, did not allow us to obtain a robust multivariate logistic regression model. In consequence, these results should be extended and replicated to a larger population before the novel biomarkers can be routinely applied in clinical practice. Furthermore, data on natriuretic peptides or troponin were not accessible for all patients. Finally, even though databases registered the expression of the parental genes in cardiac tissue, we have no confirmation about the direct secretion from the heart of these circulating circRNAs into the extracellular space. Hence, the association of circRNAs with DCM and all the interactions are putative. Further analysis should be carried out on human heart samples to confirm our results.