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Unraveling the role of miRNAs as biomarkers in Chagas cardiomyopathy: Insights into molecular pathophysiology [1]
['Heriks Gomes Ribeiro', 'Department Of Clinical', 'Toxicological Analysis', 'Federal University Of Rio Grande Do Norte', 'Natal', 'Ony Araújo Galdino', 'Karla Simone Costa De Souza', 'Antonia Pereira Rosa Neta', 'Hui Tzu Lin-Wang', 'Molecular Biology Laboratory']
Date: 2024-07
In this review, we point out miR-21, miR-146b, miR-146a, miR-155, and miR-145-5p role in the complex mechanisms of ChCM. These miRNAs have been shown as potential biomarkers for precise diagnosis, reliable prognostic evaluation, and effective treatment strategies in the ChCM.
The miR-21, miR-146b, miR-146a, and miR-155 consistently exhibited up-regulation, whereas miR-145 was down-regulated in ChCM. These specific miRNAs have been linked to fibrosis, immune response, and inflammatory processes in heart tissue. Moreover, the findings from various studies indicate that these miRNAs have the potential as biomarkers for the disease and could be targeted in therapeutic strategies for ChCM.
The search was conducted in the US National Library of Medicine MEDLINE/PubMed public database using the terms “Chagas cardiomyopathy” OR “Chagas disease” AND “microRNA” OR “miRNA” OR “miR.” Additionally, bioinformatics analysis was performed to investigate miRNA-target interactions and explore enrichment pathways of gene ontology biological processes and molecular functions.
Chagas cardiomyopathy (ChCM) is a severe form of Chagas disease and a major cause of cardiovascular morbidity and mortality. The dysregulation of the immune response leads to cardiac remodeling and functional disruptions, resulting in life-threatening complications. Conventional diagnostic methods have limitations, and therapeutic response evaluation is challenging. MicroRNAs (miRNAs), important regulators of gene expression, show potential as biomarkers for diagnosis and prognosis.
Copyright: © 2024 Ribeiro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Therefore, this review encompasses studies that have elucidated the involvement of miRNAs in the mechanisms underlying ChCM. Furthermore, it explores their function in molecular pathways involved in ChD and the potential application of miRNAs as biomarkers for accurate diagnosis, prognostic assessment, and therapeutic interventions of ChCM.
In the context of ChCM, various studies have explored miRNAs’ expression profiles in patients and identified specific miRNAs that are dysregulated in cardiac tissues [ 11 – 14 ]. These findings suggest that miRNAs could be potential biomarkers for diagnosing ChCM and monitoring disease progression [ 12 , 20 ]. Furthermore, experimental studies have demonstrated the involvement of specific miRNAs in regulating key molecular pathways implicated in ChCM, including inflammation, fibrosis, and apoptosis [ 12 – 14 , 21 , 22 ]. Consequently, targeting these miRNAs has shown promise as a potential therapeutic strategy for ChCM, with preclinical studies highlighting the beneficial effects of miRNA-based interventions in animal models of the disease [ 22 , 23 ].
Notably, miRNAs are involved in the regulation of major cellular activities, such as metabolism, differentiation, proliferation, as well as apoptosis [ 17 , 18 ] and its amount has been shown to be dysregulated in different disorders, including cardiac conditions, bacterial and viral infections, and parasitic infections [ 16 , 19 ]. These functional miRNAs can be detected in various body fluids, such as whole blood, serum, and plasma, making them potential biomarkers for the diagnosis and prognosis of the disease [ 19 ]. Further, although the exact mechanism by which miRNAs are involved in ChCM pathophysiology remains unclear, studies have revealed that the biogenesis of miRNAs implicated in cardiac physiology is affected by T. cruzi infection, and thus miRNAs could potentially serve as prospective biomarkers for ChCM [ 11 – 14 , 20 – 22 ].
Nevertheless, over the years, research has shed light on the role of microRNAs (miRNAs) in the pathogenesis and progression of ChCM [ 11 – 14 ]. These miRNAs are endogenous, conserved, small (approximately 22 nucleotides) non-coding RNA molecules that have critical roles in regulating gene expression at the posttranscriptional level. They can bind to complementary messenger RNA (mRNA) sites inhibiting protein translation or promoting mRNA decay [ 15 , 16 ].
Despite being described over a century ago, many aspects of ChD remain unresolved. Currently, conventional diagnostic and prognostic methods for ChCM primarily detect functional and structural changes in the heart [ 5 ]. Consequently, clinical and therapeutic interventions rely on the onset and progression of cardiac damage [ 6 ]. Another challenge is accurately evaluating the therapeutic response to T. cruzi, which still lacks satisfactory methods, raising concerns about the clinical management of patients [ 7 ]. Several studies have proposed biological molecules (immunological, biochemical, and molecular or nucleic acid-based biomarkers) as biomarkers for monitoring and treatment of patients with ChD, but their practical implementation has been limited, indicating there are still gaps in this area of knowledge [ 8 – 10 ].
Chagas disease (ChD) is caused by the parasite Trypanosoma cruzi and affects millions worldwide, primarily in Latin American countries [ 1 ]. While many infected individuals remain asymptomatic during the chronic phase, around 30% develop the cardiac form known as Chagas cardiomyopathy (ChCM), which is the most severe form of ChD and is a major cause of cardiovascular-related morbidity and mortality in endemic areas [ 2 , 3 ]. The dysregulation of the immune response is believed to trigger cardiac remodeling, leading to hypertrophy, fibrosis, edema, and disruptions in heart function, which can result in life-threatening arrhythmias, heart failure, thromboembolism, and sudden death [ 3 , 4 ].
Methods
Authors HGR and OAG conducted a comprehensive literature search of all original articles published up until October 15, 2023, to identify potential miRNA biomarkers in ChCM. The search was performed using the US National Library of Medicine MEDLINE/PubMed public database and included the terms “Chagas cardiomyopathy” OR “Chagas disease” AND “microRNA” OR “miRNA” OR “miR.” Additionally, MeSH terms (“Chagas Cardiomyopathy”[Mesh]) AND “MicroRNAs”[Mesh] were also included.
miRNA-target interaction and enrichment analysis A bioinformatics approach was performed to demonstrate the relationship between miRNAs and molecular and biological processes that may be related to ChCM. All analyses were performed only with the miRNAs shown in the studies, which were more prevalent and whose nucleotide sequences were conserved among the species used in the experiments and humans. The miRBase database [30] was used to query the miRNA sequences. The selection of targets was carried out at miRDB—MicroRNA Target Prediction Database [31]. Only targets with “Target Score” > = 80 were considered. Gene Ontology (GO) for Biological Processes and Molecular Functions was performed at EnrichR [32], considering terms related to cardiology and immunology. GO processes considered significant had a p-value < 0.05. The analysis and plots generated were performed using the software RStudio Version 2023.06.0+421 with Packages ggplot2 Version 3.4.2 [33] and Circlize Version 0.4.15 [34].
miRNA for detection and progression Gòmez-Ochoa and colleagues [11] discovered a striking correlation between the expression levels of circulating miR-223-5p by qRT-PCR in serum samples. They correlated both echocardiographic and laboratory biomarkers of myocardial injury in a group of 74 patients with ChCM. Lower levels of miR-223-5p were associated with the worsening of ChCM, potentially attributed to signaling pathways associated with receptor tyrosine kinases. These findings provide insights into the mechanistic role of miR-223-5p in the progression of this condition. In another study conducted by Ballinas-Verdugo and colleagues [12], the overexpression of inflammatory microRNAs miR-21, miR-146a, and miR-155 were evaluated in heart tissue, plasma, and plasma extracellular vesicles from mice infected with the Mexican TcI Ninoa strain. The qRT-PCR was performed to assess miRNAs in the acute and chronic phases of infected mice. The study also identified 23 functional pathways associated with T. cruzi infection. Furthermore, bioinformatics analysis identified 11 genes, including down-regulated SMAD family member five, which is a mediator in the transforming growth factor-beta (TGF-β) signaling pathway. The researchers emphasized that miR-146a could be a potential noninvasive biomarker for the early detection of ChD. Nonaka and colleagues (2019) [13] assessed 48 subjects, including individuals with the cardiac form of ChD presenting left ventricular dysfunction, indeterminate individuals without symptoms of ChD, and healthy controls. The qRT-PCR was applied to evaluate miRNA expression in exosomes derived from serum and plasma samples. The findings revealed significant alterations in the expression levels of circulating miRNAs in ChD, some of which correlated with the expression profile in cardiac tissue. Notably, elevated levels of miR-19a-3p, miR-21-5p, and miR-29b-3p in circulation were associated with cardiac injury and the severity of ChD. The authors further validated their finding of miRNAs through in vitro models of cardiac fibrosis using TGF-1-stimulated cardiac fibroblast cells and hypertrophy myocardial model using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) stimulated with endothelin-1. Elevated expression of miR-19a-3p, miR-21-5p, miR-29b-3p, and miR-199b-5p was observed in hiPSC-CM, while miR-21-5p was up-regulated in cardiac fibroblasts. These results provide insights into the potential roles of specific miRNAs in the pathogenesis and progression of ChCM. Linhares-Lacerda and colleagues [14] conducted a miRNA expression level analysis by qRT-PCR using serum samples from 40 patients, including those indeterminate individuals without ChD symptoms and non-infected controls. Interestingly, the elevated levels of circulating miR-208a were identified only in the indeterminate form of ChD. It suggests that circulating miR-208a could be a potential biomarker of ChD when it is under control. Rego and colleagues [26] conducted a study using cardiomyocytes, epithelial tissue, and macrophages from humans. The cells were infected with T. cruzi, and analyses were carried out 24 h after infection. miRNA sequencing was conducted, and robust bioinformatics analyses revealed that macrophages were more responsive to infection than other cell types. In the context of results among different tissue types, cardiomyocytes showed a higher degree of infection in the early stages than other cells. As demonstrated in previous studies, miRNA-146a is strongly associated with parasite infection. Furthermore, the study revealed the up-regulation of miR-1246 and miR-708, which have not yet been reported in the context of infection. Functional analyses did not reveal a direct relationship with targets already recognized in previous studies. However, further studies are needed to understand the role of these miRNAs and their potential use as biomarkers in ChCM.
miRNAs involved in cardiac remodeling ChCM presents a worse prognosis compared to other heart diseases. In a study by Ferreira and colleagues [24], the differences in miRNA expression profiles between DCM and ChCM were evaluated. Human left ventricular free wall heart tissue samples were obtained from end-stage heart failure patients, including ChCM samples (n = 10), DCM samples with negative serology for ChD and ischemic disease (n = 6), and control samples from healthy hearts (n = 4). Ingenuity Pathways Analysis (IPA) was utilized for the Target Prediction of differentially expressed genes (DEGs) and their relationship with the analyzed miRNAs. Five miRNAs (miR-1, miR-133a-2, miR-133b, miR-208a, and miR-208b) showed differential expression between ChCM samples and controls. Specifically comparing ChCM and DCM, miR-1, miR-133a-2, and miR-208b were down-regulated in ChCM. These miRNAs seem to interact with essential genes involved in the heart’s electrical conduction, such as Serine/Threonine Kinase 1 (AKT), Cyclin D1 (CCND1), Interferon (IFN), Growth hormone, Collagen Type 1, and Nuclear Factor kappa B (NF-κB). Notably, miR-208a and miR-208b showed significant positive associations (p-value = 0.0007) when comparing ChCM and controls, indicating their specific roles in heart muscle regulation, particularly GATA Binding Protein 4. Continuing the pioneering studies published by Ferreira and colleagues [24], Navarro and colleagues [22] investigated the role of miRNAs in disease progression following T. cruzi infection, which is associated with various cardiovascular disorders. Using a qRT-PCR array, they screened 641 rodent miRNAs in heart samples of mice during acute infection with the Colombian T. cruzi strain. Seventeen miRNAs were significantly dysregulated at all 3 analyzed time points postinfection. Among these, miR-146b, miR-21, miR-142-3p, and miR-142-5p were up-regulated, while miR-145-5p and miR-149-5p were down-regulated, and their expression correlated with parasitemia and the maximal heart rate-corrected QT (QTc) interval. Interestingly, although this study focused on the acute phase of experimental ChD, some miRNAs (miR-133, miR-208) were found to be down-regulated at 45 days postinfection, consistent with previous reports in the hearts of chronic Chagas patients. In a study by Laugier and colleagues [20], an integrative genome-wide analysis was conducted to understand the role of miRNAs in the pathophysiology of ChCM. Tissue samples from the left ventricular wall of patients with ChCM undergoing heart transplantation (n = 8) and of the organ donors (control group) (n = 4) were used for miRNA analysis. Interaction analyses between DEGs and differentially expressed miRNAs (DEMs) revealed relationships in important biological processes related to ChCM, including inflammation, interferon gamma-induced genes, fibrosis, extracellular matrix, and hypertrophy processes. Five miRNAs (hsa-miR-125b-5p, hsa-miR-15a-5p, hsa-miR-296-5p, hsa-miR-29c-3p, and hsa-miR-103a-3p) showed clear correlations in the regulation of these processes, suggesting their potential as targets for further studies and therapeutic candidates in the context of ChCM.
miRNAs for therapeutic response The first evidence of miR-145-5p down-regulation and miR-146b-5p up-regulation in acute Chagas’ heart disease was reported by Navarro and colleagues [22]. Building on this, Farani and colleagues [23] conducted a study to explore the effects of interventions with the trypanosomicidal drug Benznidazole (Bz) alone or in combination with Pentoxifylline (PTX) on parasite load and the expression of miR-145-5p and miR-146b-5p. They utilized the H9C2 rat cardiomyoblast cell line infected with the Colombian T. cruzi strain as a model to investigate the interplay between the parasite and host cells. The Bz at concentrations of 3 μm and 10 μm, 48 h after infection, reduced parasite load but did not affect the levels of miR-145-5p and miR-146b-5p by qRT-PCR analyzed. The addition of PTX did not interfere with the parasite control efficacy induced by Bz. However, the combined treatment of Bz + PTX resulted in decreased levels of both miRNAs, with expression levels like those observed in non-infected H9C2 cells. Furthermore, using mimic/inhibitor systems for miR-145-5p and miR-146b-5p before infection of H9C2 cells, a reduction in parasite load was observed 72 h postinfection. When the mimic/inhibitor systems were applied 48 h after infection, all systems except the miR-146b-5p inhibitor resulted in a reduction in parasite load. These findings suggest that miR-145-5p and miR-146b-5p may play a role in controlling signaling pathways crucial for interacting with the parasite and host cells [23]. Therefore, further investigation of these miRNAs is warranted as potential biomarkers for parasite control and tools to identify therapeutic adjuvants for etiological treatment in ChD. A recent study conducted by Silva Grijó Farani and colleagues [25] also investigated the expression profiles of 752 miRNAs by qRT-PCR array in the cardiac tissue of mice with chronic T. cruzi infection treated with Bz, PTX, or a combination of both (Bz + PTX). After 150 days of infection, the Bz + PTX-treated group showed 58 DEMs associated with key signaling pathways related to cellular growth, tissue development, cardiac fibrosis, damage, and cell death. Similarly, the Bz-treated group exhibited 68 DEMs involved in pathways such as cell cycle, cell death and survival, tissue morphology, and connective tissue function. Notably, the combined therapy restored the overexpression of miR-146b-5p, miR-196c-5p, and miR-210-3p, and the underexpression of miR-497a-5p, which play roles in regulating genes associated with heart damage, cell death, and fibrosis pathways. Further analysis revealed that miR-146b-5p directly targeted the mRNA of gene IL10 (Interleukin 10), TNF (Tumor Necrosis Factor), MMP9 (Matrix Metallopeptidase 9), ERK (Mitogen-Activated Protein Kinase 1), and JNK1 (Mitogen-Activated Protein Kinase 8). In contrast, miR-196c-5p targeted ANXA1 (Annexin A1) and BAK1, among others. These findings emphasize the complexity of ChCM and the central role of miR-146b-5p in cardiotoxicity networks, which was validated as a potential therapeutic target. The study also demonstrated that the association of Bz and Bz + PTX therapies could reverse the up-regulation of miR-146b-5p in the infected group, highlighting their potential for mitigating ChCM progression.
miRNAs as therapeutic targets Nonaka and colleagues (2021) [21] proposed miR-21 silencing as a possible therapy for ChCM. Through the combination of PCR array processes and in silico analyses, they identified that miR-21 was up-regulated in cardiac tissue samples from mice and human serum. In addition, functional studies showed that miR-21 was associated with important regulatory pathways of collagen expression (TGFβ1). Twelve C57Bl/6 mice were used in the locked nucleic acid (LNA)-anti-miR-21 inhibitor promoted efficacy test. Mice were infected by intraperitoneal injection of 1,000 trypomastigotes of the Colombian T. cruzi strain. Heart samples from chronic chagasic mice (n = 8) and non-infected controls (n = 4) were evaluated after 6 months. The results showed the reduction of miR-21 expression in the cardiac tissue of mice treated with anti-miR-21 compared to untreated mice. Another study using H9C2 cells infected with the T. cruzi Berenice 62 strain and assessed the expression of specific miRNAs (miR-16-5p, miR-let7f-2-3p, miR-26b, miR-3586-3p, miR-190b) by qRT-PCR [27]. Subsequently, luciferase reporter assays were conducted, explicitly targeting miR-190b with a transiently introduced no-miR-190b inhibitor. The results demonstrated a correlation between miRNA-190b and decreased cellular viability rates, achieved by negatively modulating phosphatase and tensin homolog (PTEN) protein expression in T. cruzi-infected cells. PTEN is a tumor suppressor and negative regulator of PI3K signaling and plays a crucial role in hydrolyzing PIP3 to phosphatidylinositol-4,5-bisphosphate. Previous studies have shown that PTEN is reduced during hypertrophic cardiomyopathy and is associated with remodeling in cardiomyocytes [35]. This study sheds light on the modulation of cellular behavior by parasite infection, ensuring the survival of T. cruzi. In the study of Monteiro and colleagues [27], the reduced expression of PTEN, favored by the increase in expression levels of 5 miRNAs, including miR-190b, during the early stages of T. cruzi Be-62 infection, underscores its involvement in PTEN regulation and calls for further investigation.
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