3.1. MicroRNAs in CVD and Hypertension
MiRNAs are non-coding RNAs endogenous, highly conserved, single-stranded, small (~22 nucleotides), stable and ubiquitously detectable, including in the circulation of an organism, both in plasma and serum (Figure 2
]. MiRNAs regulate gene expression post-transcriptionally by binding to the 3’-untranslated regions of target mRNAs [21
]. Each miRNA potentially targets many unique mRNAs, inhibiting translation and/or inducing degradation of the target mRNA depending on the number and accessibility of binding sites. Higher complementarity between the miRNA and its target(s) and greater levels of mRNA inhibition or degradation are observed [16
]. Changes in miRNA expression levels have been associated with several CVDs including hypertension, atherosclerosis, CAD, myocardial infarction (MI), HF and cardiac arrhythmias, suggesting their potential use as therapeutic targets and diagnostic and prognostic biomarkers [22
]. Several review articles have highlighted the main findings on miRNA expression and associations with CVDs [15
]. In this review, we will provide a few examples to illustrate their importance.
Recent studies have described approximately 50 miRNAs associated with essential hypertension and over 30 with HF and MI, with many of those miRNAs described as promising biomarkers [23
]. The renin-angiotensin system is a balanced network that regulates blood pressure with multiple miRNAs involved. The downregulation of miR-34b, miR-361-5p, miR-362-5p, and miR-181a and the upregulation of miR-34c-5p, miR-449b, miR-571, miR-765, miR-483-3p, miR-143/145, miR-126, miR-196a, miR-132, miR-212, miR-451, and miR-21, independently or as a group, impact this system and seem to cause an increase in blood pressure [25
]. MiR-21 is expressed in many cell types related to cardiovascular health, including vascular smooth muscle cells (VSMC), vascular endothelial cells, myocardial cells, cardiac fibroblasts and blood [26
]. Its expression is closely related to the development and progression of HT and related to target organ damage, including the regulation of the renin angiotensin system, inflammatory cytokines and endothelial function [27
]. In addition to the miR-21 role in HT development, research into cardiac dysfunction post MI suggests its critical involvement in cardiac fibroblast activation and cardiac fibrosis via the TGFβ/Smad7 signalling pathway [28
Elevated plasma levels of miR-1, miR-133a, miR-499 and miR-208a have been described in acute MI, highlighting its potential use as a biomarker for early diagnosis [29
]. In addition, miR-19a/19b are upregulated in heart failure patients after MI and the delivery if its mimics to the heart increases cardiac proliferation and regeneration, indicating a possible compensatory mechanism in response to stress [30
Higher levels of miRNA-29a are strongly correlated with hypertrophic cardiomyopathy and fibrosis of the heart [31
]. MiRNAs have also been described in the progression and regression of atherosclerosis, via the regulation of lipoprotein homeostasis. The downregulation of miR-145 encourages plaque formation in the vasculature of patients through decreased VSMC differentiation [32
]. One of the first studies to use miRNA for potential medical intervention in atherosclerosis, treated ApoE−/− mice, which are pre-disposed to developing atherosclerosis, with a lentivirus, aiming to increase the expression of miR-145. The upregulation of miR-145 after lentivirus treatment resulted in a decrease in plaque formation in the vasculature of the ApoE−/− mice [33
Another role for miRNAs is their potential involvement in the regulation of angiotensin-converting enzyme 2 (ACE2). ACE2 inactivates the BP regulating angiotensin II (Ang II) by cleaving it to the Ang 1–7 products [34
]. Reduced expression levels of ACE2 have been reported in hypertension [36
]. More intriguing is that it also acts as the tissue receptor for coronavirus (COVID-19) [35
]. A study by Liu et al. found that miR-200c-3p directly targets the 3′-untranslated region of ACE2 and downregulates its protein expression [39
]. These observations have interesting implications for the development of novel miRNA therapeutics for both hypertension and COVID-19.
Importantly, recent studies also highlight the relevance of miRNAs in modulating the levels of ACE2 in other CVD. MiRNA let-7b is upregulated in hypoxic pulmonary hypertension which decreases levels of ACE2. The in vitro decrease of let-7b eased the development of hypoxic pulmonary hypertension [40
]. Patients with chronic kidney disease and on haemodialysis have high levels of serum miR-421 and low levels of Ang 1–7 products and ACE2, which have been described as being able to contribute to the development of atherosclerosis [41
]. Lastly, miR-125b is a negative regulator of ACE2 after high glucose treatment. The downregulation of ACE2 was hindered after knocking down miR-125b in human kidney cells, which also reduced reactive oxygen species and apoptosis after high-glucose treatment. The interactions between miR125b and ACE2 present a potential therapeutic target in diabetic nephropathy [42
Despite ongoing investigations into miRNAs and CVD, its incorporation into clinical practice has not occurred. This is mainly occurring due to the lack of an easy-to-handle, fast, reliable, and inexpensive method to determine miRNAs levels. However, many miRNAs are currently being used as part of clinical trials, which may lead to early diagnosis, prevention and treatment.
3.2. Long Non-Coding RNAs and Their Involvement in CVD and Hypertension
LncRNAs are greater than 200 nucleotides in length, tissue specific and poorly conserved across species. They play a role in transcriptional and post-transcriptional gene regulation and mRNA translation, being involved in epigenetic modifications, the modulation of alternative splicing and transcription, or interacting with mRNAs and proteins in the cytoplasm to regulate gene expression (Figure 2
Similarly to miRNAs, a large number of lncRNAs has been described to be involved in the critical regulation of several cardiac disorders, highlighting their role in the development and progression of CVD [25
]. Importantly, expression of genes such as ACE2 are not only regulated by miRNAs, but also lncRNAs. ALT1 is a lncRNA downregulated in hypoxia induced, growth arrested human umbilical vein endothelial cells and a direct target of ACE2 [43
LncRNA growth arrest-specific 5 (GAS5) was shown to regulate VSMC and endothelial cells (EC) in animal models [44
]. Vascular remodelling is strongly correlated with the dysfunction of VSMC and EC, suggesting a role of GAS5 to the development of essential hypertension [44
]. Another study, using microarray analysis, identified 145 differentially expressed lncRNAs in the ipsilateral renal cortex when comparing spontaneously hypertensive and normotensive rats [45
A cluster of lncRNAs from the Myh7 gene was identified as functionally significant in cardiac hypertrophy in mice [46
]. Myh7, a myosin heavy chain associated RNA transcript (Mhrt) was irreversibly reduced after the induction of cardiac hypertrophy through transverse aortic surgery (TAC). The overexpression of Mhrt reduced cardiac hypertrophy and fibrosis leading to an improvement in cardiac function was observed when compared to TAC operated mice without reactivated Mhrt [46
The lncRNA cardiac hypertrophy associated transcript, or CHAST, is upregulated in cardiomyocytes of TAC operated mice and involved in cardiomyocyte hypertrophy. CHAST is also upregulated in patients with cardiac hypertrophy and cardiomyocytes derived from human embryonic stem cells. Research shows that the overexpression of CHAST induces cardiomyocyte hypertrophy in cell and animal models. Meanwhile, its silencing prevents TAC induced cardiac remodelling with no toxicological side effects. Interestingly, pleckstrin, the opposite strand of CHAST, inhibits cardiomyocyte autophagy and hypertrophy, suggesting that CHAST could be a potential target to prevent cardiac remodelling, while indicating a general role of lncRNAs in heart diseases [47
The lncRNA myocardial infarction associated transcript (MIAT) has been identified as promoting cardiac fibrosis and remodelling after MI [48
]. Cardiac fibrosis presents after MI and cardiac remodelling of the infarct region is key in sustaining myocardial integrity, through preventing wall rupture during the healing process. Conversely, fibrosis caused by cardiac remodelling increases cardiac stiffness and impairs cardiac function that can lead to HF [49
]. Furthermore, other studies revealed that MIAT acts as pro-hypertrophic in cardiomyocytes through sponging anti-hypertrophic miR-93 and miR-150 [50
]. The oxygen deficiency caused by MI leads to a significant loss in viable cardiomyocytes by necrotic cell death and apoptosis. The lncRNA, mitochondrial dynamic related lncRNA (Mdrl) and cardiac apoptosis-related lncRNA (Carl) are both reduced after MI [52
]. Increased expression of Mdrl and Carl inhibited cardiomyocyte apoptosis through reduction in miRNAs miR-361 and miR-539, resulting in reduced infarct sizes. All these studies suggest that lncRNAs are critical regulators of cardiac fibrosis and cardiomyocyte survival in hypertrophic and infarcted hearts by gene regulation interference, through interactions with other ncRNAs such as miRNAs.
A study by Wang et al. [54
] identified the lncRNA cardiac hypertrophy associated epigenetic regulator (Chaer) as essential for the development of cardiac hypertrophy in a mouse model of pressure overload induced failing hearts. Interestingly, Chaer knockdown significantly supressed chemically induced hypertrophy by phenylephrine, but did not interfere with myocyte morphology at basal level. Chaer directly interacts with polycomb repressor complex 2 (PRC2), which inhibits downstream genes involved in cardiac hypertrophy. The interaction between Chaer and PRC2 is briefly induced post hormone or stress stimulation and this interaction is a requirement for the epigenetic reprogramming that activates genes involved in cardiac hypertrophy. The inhibition of Chaer expression in the heart prior to the onset of pressure overload decreases cardiac hypertrophy and dysfunction, but is not impacted in post-stressed hearts [54
3.3. Circular RNAs: What Are They and How Do They Function?
CircRNAs are abundant, underexplored ncRNAs. Recent studies revealed that large numbers of circRNAs are endogenous, highly conserved and stable in mammalian cells and prevalent in disease states (Figure 2
]. Although mRNA and circRNAs both originate from precursor-mRNAs, they are formed differently, giving them unique characteristics. mRNAs are formed by RNA splicing where introns are removed, and certain exons are included or excluded to create alternative coding mature mRNAs. This process creates linear mRNAs with exposed poly(A) tails. This characteristic leaves them prone to degradation by RNases [55
]. Meanwhile, circRNAs are formed by back-splicing, promoting the circularization process where exons and/or introns converge onto each other, potentially protecting them from degradation and conferring a half-life of approximately 48 h, around five times more stable than linear mRNAs (Figure 3
The definitive function of circRNAs still remains unclear. It has been proposed that circRNAs regulate the expression of linear mRNA transcripts both directly, via the competition with the splicing machinery [57
]; and indirectly, acting as sponges to miRNAs due to the presence of multiple binding sites, allowing them to interact with and sequester cellular miRNAs preventing the performance of their roles on post-transcriptional regulation (Figure 4
Importantly, circRNAs make up 1% of total RNA being expressed widely in various cell types and may have a regulatory function in human disease, with a pivotal role in the initiation and progression of various types of biological processes [58
], potentially acting as a biomarker for the discovery and investigation on the progression of disease. However promising, research to identify and characterize circRNAs has mostly been performed using bioinformatics and in silico approaches and a limited number of studies have investigated their function in situ or in vivo to establish their involvement in disease.
The properties described above and promising research in the field of cancer genomics using circRNAs as biomarkers is encouraging and should be explored and translated into cardiovascular genomics research. This is extremely relevant, as conventional methods for controlling risk factors and initiating early treatment in CVD intervention have often led to poor prognosis. Current biomarkers usually detect the disease at later stages of development, increasing the need for the discovery of new biomarkers for prevention or at early onset of disease. This further highlights the benefits of using circRNAs as potential biomarkers in CVD (Table 1
Emerging evidence of circular RNAs in cardiovascular disorders has demonstrated differential expression in both healthy and diseased hearts [60
]. However, the relevance of circular RNAs to the cardiovascular system remains poorly characterised, and an improvement in understanding of circRNA involvement in CVD will form a basis for the development of these RNAs as biomarkers for discovery, prediction and therapeutic agents. Importantly, the combination of genetic sequencing and bioinformatics discovery has enabled the identification of many novel circRNAs.