MiRs and congenital heart diseases

Congenital heart defects account for ∼40% of prenatal deaths and more than 20% of deaths in the first month of life.Reference Trojnarska, Grajek, Katarzynski and Kramer ⁷ A complete cure of a congenital heart defect in childhood is exceptional, and with increasing life expectancy the population of adults with clinical manifestation of congenital heart diseases continues to expand, reaching up to 90% of children born with congenital heart diseases.Reference Khairy, Ionescu-Ittu, Mackie, Abrahamowicz, Pilote and Marelli ⁸ Among adults in the year 2000, the median age of the population with congenital heart diseases was 40 years, with a median age of 29 years in those with severe disease versus 42 years in those with other congenital heart diseases.Reference Marelli, Mackie, Ionescu-Ittu, Rahme and Pilote ⁹

MiRs are known now to play central roles as governors of gene expression during cardiovascular development,Reference Bruneau ¹⁰ involving the integration of multiple cell lineages into the three-dimensional organ and its connection to the vascular system.Reference Chen and Wang ¹¹ The important roles of miRs in cardiogenesis and early embryonic patterning processes are evidenced by the rapid increase in detectable miRs in tissues derived from all three germ layers.Reference Ivey, Muth and Arnold ¹² Such roles are further confirmed by gain and loss of function experiments in mice showing that aberrant expression of selective miR produce defects.Reference Callis, Chen and Wang ¹³

MiR-1 was the first miR shown to regulate fundamental aspects of heart development.Reference Zhao, Samal and Srivastava ¹⁴ Overexpression of miR-1 in the embryonic heart inhibits cardiomyocyte proliferation and prevents expansion of the ventricular myocardium, causing lethality due to deficiency of cardiomyocytes and insufficient muscle mass.Reference Zhao, Samal and Srivastava ¹⁴ Consistent with this, development of Xenopus hearts is also blocked by injecting embryos with miR-1.Reference Chen, Mandel and Thomson ¹⁵ Targeted deletion of miR-1-2 in mice resulted in 50% embryonic lethality, largely due to ventricular septal defects, whereas the surviving mutant mice also died at a later stage because of conduction system defects.Reference Zhao, Ransom and Li ¹⁶

Conditional deletion of Dicer, the enzyme required for miR processing, causes mouse embryos to die from cardiac failure by embryonic day 12.5 (E12.5) because of underdeveloped ventricular myocardium.Reference Chen, Murchison and Tang ¹⁷ Disturbance of neural crest cell migration into the derivatives of the pharyngeal arches and pouches can account for many of the developmental defects. Intriguingly, phenotypic overlap between genetic disorders in cardiovascular and neuronal-craniofacial defects, including DiGeorge Syndrome, Noonan syndrome, LEOPARD syndrome, cardio-facio-cutaneous syndrome, and Costello syndrome, has been described.Reference Roberts, Allanson and Jadico ^{18 ,} Reference Perez and Sullivan ¹⁹ The targeted deletion of Dicer in neural crest cells led to severe craniofacial and cardiovascular defects, which are reminiscent of features of human congenital neuro-craniofacial-cardiac defects.Reference Huang, Chen and Regan ²⁰

MiR-133a-1/miR-1-2 and miR-133a-2/miR-1-1 genes are expressed throughout the ventricular myocardium and interventricular septum from E8.5 until adulthood.Reference Zhao, Samal and Srivastava ^{14 ,} Reference Liu, Williams and Kim ²¹ Mice lacking either miR-133a-1 or miR-133a-2 do not display obvious cardiac abnormalities, whereas deletion of both miRs results in late embryonic and neonatal lethality due to ventricular septal defects and chamber dilatation.Reference Liu, Bezprozvannaya and Williams ²² Similar cardiac abnormalities are observed by targeted deletion of the miR-17∼92 cluster.Reference Ventura, Young and Winslow ²³ MiR-196a was found in foetal human heart samples at a gestational age of 12–14 weeks.Reference Thum, Galuppo and Wolf ²⁴ This miR regulates HOXB8-Shh signalling, which is required throughout cardiac septation, outflow tract morphogenesis, and valve formation.Reference Goddeeris, Rho and Petiet ²⁵

Just recently, miRs have been explored in one of the congenital cyanotic heart diseases.Reference O'Brien, Kibiryeva and Zhou ²⁶ Expression studies of miRs in the right ventricular myocardium of children with non-syndromic tetralogy of Fallot showed a significantly altered expression of 61 miRs. Potential targets of the altered miRs are gene networks important for cardiac development.Reference O'Brien, Kibiryeva and Zhou ²⁶

Comprehensive miR profiling in human foetal single-ventricle cardiac tissues revealed 48 differentially expressed miRs of which 38 were downregulated and 10 were upregulated in comparison to control cardiac tissue.Reference Yu, Han, Bai, Zhu, Pan and Guo ²⁷

Dysregulation of miRs has also been reported with syndromic congenital heart diseases such as trisomy 21 (Down syndrome), the most common genetic cause of congenital heart defects.Reference Kuhn, Nuovo and Martin ²⁸ A total of five miRs – miR-99a, let-7c, miR-125b-2, miR-155, and miR-802 – were identified on human chromosome 21, and were found to be overexpressed in the heart of afflicted patients who contained the extra Hsa21 chromosome.Reference Latronico, Catalucci and Condorelli ²⁹ DiGeorge syndrome, which in many patients leads to congenital heart disease, is caused by a deletion of the DiGeorge syndrome critical region 8 on chromosome 22 (22q11.2), which encodes a component of the RNA-induced silencing complex. The fact that this syndrome results from haplo-insufficiency of this locus raises the possibility that perturbation of miR expression could contribute to the gene dosage sensitivity of this disease by impacting numerous miR targets.Reference Han, Lee, Yeom, Kim, Jin and Kim ³⁰ In conclusion, it is possible that lethal cardiovascular diseases related to both of the major genetic backgrounds of syndromic congenital heart diseases, Down and DiGeorge Syndrome, are caused by dysregulated expression of specific miRs including miR-99a, let-7c, miR-125b-2, miR-155, miR-802, and RNA-induced silencing complex.

MiRs and cardiometabolic clusters

Childhood obesity is a major concern because of its close association with hypertension, dyslipidaemia, and type 2 diabetes mellitus. The worldwide prevalence of childhood obesity increased from 4.2% in 1990 to 6.7% in 2010. If this trend is to continue, the prevalence is expected to reach 9.1% by 2020.Reference de Onis, Blossner and Borghi ³¹ With the rise in obesity rates, metabolic syndrome has become prevalent among children worldwide,Reference Ferreira, Oliveira and Franca ³² which is one of the most serious challenges to global health in the modern world. Obesity and type 2 diabetes mellitus are major risk factors for coronary heart disease, the leading cause of death in the West.Reference Lloyd-Jones, Adams and Carnethon ³³ It is estimated that the prevalence of coronary heart disease in the United States will increase by 16% by the year 2035 – a significant component attributable to increased type 2 diabetes mellitus and adolescent obesity.Reference Bibbins-Domingo, Coxson, Pletcher, Lightwood and Goldman ³⁴ This will place a major and perhaps insurmountable burden on our healthcare systems, and preventive measures, early diagnosis, and early intervention are urgently needed. Obese children develop early-onset atherosclerotic lesions and clustering of metabolic abnormalities that persist during adulthood.Reference Akgun, Dogan and Akbayram ^{35 –} Reference Hayman, Meininger and Daniels ³⁸ This renders childhood obesity as a consistent predictor of adult heart disease and hypertension in both young and old.Reference Baker, Olsen and Sorensen ³⁹ Much evidence now indicates aberrant genetic components including miRs that cause enhanced predisposition of some individuals to obesity and type 2 diabetes, and treatment strategies based on these are under development.

MiRs have been shown to have regulatory roles in glucose and lipid metabolism and many of the steps leading to obesity, including adipocyte development, proliferation, differentiation, insulin action, and fat metabolism.Reference Heneghan, Miller and Kerin ^{40 –} Reference Kim, Kim and Lee ⁴⁴

In vitro cell studies showing that different miRs increase at different stages of adipocyte development suggest key roles for miRs in the stage-specific regulation of adipogenesis. MiR-21 increased transiently during early adipogenic differentiation of human multi-potent mesenchymal stem cells,Reference Kim, Hwang, Bae and Jung ⁴⁵ while increased miR-20 levels were reported in mature adipocytes.Reference Esau, Kang and Peralta ⁴⁶ Overexpression of miR-210 promotes lipid droplet formation and adipocyte hypertrophy in 3T3-L1 cells.Reference Qin, Chen and Niu ⁴² Similar effects were observed by downregulation of miR-27b during adipocyte differentiation.Reference Karbiener, Fischer and Nowitsch ⁴⁷ Adipocyte proliferation is further promoted by miR-15 through fine-tuning of Dlk1, whereas miR-15a inhibition appears to reduce pre-adipocyte size.Reference Andersen, Jensen and Schneider ⁴⁸

MiR-103 and the closely related miR-107 are upregulated in the liver of obese mice, and treatment of mice with miR-103/107 antagomiRs was shown to reduce obesity.Reference Trajkovski, Hausser and Soutschek ⁴⁹ MiR-103 is also upregulated during differentiation of human pre-adipocytes, and its levels are enhanced during adipogenic stimuli, for example, in response to increased triglycerides. One of the targets of miR-103/107 is caveolin-1, and downregulation of caveolin-1 expression by miR-103/107 results in decreased insulin sensitivity by inhibition of the insulin receptor, depressed AKT activity, and decreased glucose uptake. Other targets pathways of miR-103/107 include enzymes of acetyl-CoA and lipid metabolism.Reference Wilfred, Wang and Nelson ⁵⁰ These miRs are attractive candidates for the treatment of hyperlipidaemia and obesity possibly through the use of liver-directed anatgomiRs. Similar to all such approaches, there may be off-target consequences of such treatments, which may cause adverse side effects; such safety issues will need to be thoroughly tested in appropriate preclinical models.

MiRs have been identified that enhance or inhibit growth and differentiation of adipose. For example, overexpression of miR-27a in pre-adipocytes suppresses adipocyte differentiationReference Lin, Gao, Alarcon, Ye and Yun ⁵¹ possibly by repression of PPARγ, an established transcriptional factor for adipogenic genes.Reference Kim, Kim and Lee ^{44 ,} Reference Wang, Li and Guan ⁵² MiR-27a expression is depressed in mature adipocytes from obese mice compared with lean mice, suggesting that miR-27a downregulation is required for adipocyte hypertrophy.Reference Kim, Hwang, Bae and Jung ⁴⁵ Another potential inhibitor of adipogenesis is miR-448, which targets and represses transcription factor Krueppel-like factor 5, a nuclear protein that binds the epidermal growth factor response element.Reference Kinoshita, Ono and Horie ⁵³

In addition to obesity, hyperglycaemia is another major component of the metabolic syndrome that is also under significant regulation by miRs (reviewed in).Reference Natarajan, Putta and Kato ⁵⁴ MiRs significantly regulate the production and secretion of insulin, while simultaneously affecting the sensitivity of its target tissues.Reference Poy, Spranger and Stoffel ⁵⁵ Specifically, pancreatic islet-specific miR-375 along with miR-124a and let-7b play key roles in blood glucose homeostasis through regulation of β-cell function, particularly exocytosis of insulin-containing vesicles. MiR-124a and let-7b are both abundantly expressed in pancreatic islet β-cells. MiR-30d influences insulin transcription and protects β-cell functions from impairment by proinflammatory cytokines by targeting mitogen-activated protein 4 kinase 4.Reference Tang, Muniappan, Tang and Ozcan ⁵⁶ The resultant hyperinsulinaemia causes an increased rate of fat storage and deposition in organs and tissues. MiR-33a and -b regulate three of the major metabolic pathways involved in the risk for metabolic syndrome. In concert with their host genes, the sterol-regulatory element-binding protein transcription factors, miR-33a and -b, balance cholesterol metabolism, fatty acid oxidation, and insulin signalling.Reference Terán-García and Bouchard ⁵⁷ Treatment of metabolic syndrome and decreasing its prevalence is the ultimate intermediate goal in the process of preventing coronary heart disease, which renders miR-33a and -b of special interest for further research.

Metabolic derangements including insulin resistance, hyperlipidaemia, and hyperglycaemia are accompanied by dysregulation of specific sets of miRs, and these conditions in turn trigger dysregulation of secondary miRs with targets that lead to obesity, metabolic syndrome and ultimately increased risk of cardiovascular disease. These miRs work by positive and negative regulation of multiple genes and are becoming attractive targets for global suppression of metabolic syndrome.

MiRs and heart failure

Heart failure is a major public health problem, affecting nearly 23 million people, and accounts for 5% of all medical hospital admissions and 2% of global health spending worldwide. Heart failure among accounts for at least 50% of referrals for paediatric heart transplantation.Reference Boucek, Edwards, Keck, Trulock, Taylor and Hertz ⁵⁸ The largest heart failure burden comes from children with congenital malformations. It has been estimated that 15% to 25% of children who have structural heart disease develop heart failure.Reference Kay, Colan and Graham ⁵⁹ The involvement of miRs in the pathogenesis and progression of heart failure is further supported by a recent review,Reference Tijsen, Pinto and Creemers ⁶⁰ explaining the role of miRs in myocyte hypertrophy, cardiomyocyte apoptosis, interstitial fibrosis, reduced capillary density, and activation of the immune system. This review documents the growing evidence that miRs contribute to pathological remodelling of the heart by regulating the expression of target genes that are involved in fibrosis, endothelial cell function, angiogenesis, and inflammation. Although some miRs have very specific functions in one cell type – for example, miR-126 in endothelial cells, other miRs are more ubiquitously expressed and regulate gene expression in multiple cell types.Reference Tijsen, Pinto and Creemers ⁶⁰

Recent studies comparing miR expression profiles from failing, non-failing, and foetal human hearts found that reactivation of a foetal miR program may be a feature of gene expression defects in the failing human heart.Reference Thum, Galuppo and Wolf ²⁴ Specific miRs are consistently found to be aberrantly expressed in the myocardium of heart failure patients and reveal a signature pattern of expression. Among these are miR-1, miR-29, miR-30, miR-133, and miR-150 that are downregulated in heart failure patients, whereas miR-21, miR-23a, miR-125, miR-146, miR-195, miR-199, and miR-214 were upregulated.Reference Martinez, Patkaniowska, Urlaub, Luhrmann and Tuschl ^{61 –} Reference Matkovich, Van Booven and Youker ⁶³ Interestingly, most of the constitutively down- and upregulated miRs during heart failure are similarly down- and upregulated in cardiomyocyte-specific Dicer knockouts, suggesting a normal high-level expression in cardiomyocytes. Dicer knockout hearts have severely depressed amounts of contractile proteins and consequent contractile insufficiency. Therefore, miRs have clear and essential roles in myocardial development and maturation, and defective expression of these miRs may contribute significantly to the origin and progression of congestive heart failure.Reference Topkara and Mann ⁶⁴ In terms of mechanism of action, there is emerging evidence that many of the identified miRs regulate the expression levels of genes that govern the process of adaptive and maladaptive cardiac remodelling.Reference Topkara and Mann ⁶⁴ For example, miR-17∼92 has been reported to target connective tissue growth factor that, in the heart, is associated with adverse remodelling during heart failure.Reference Ernst, Campos and Meier ^{65 ,} Reference Schellings, Vanhoutte and van Almen ⁶⁶ Connective tissue growth factor is a matricellular protein with roles in many biological processes, including cell adhesion, migration, and proliferation, and with a critical role in regulating inflammation and fibrosis, disease suggests a further role of inflammation-associated miRs in the pathogenesis of heart failure. This suggestion is further supported by increased macrophage-derived miR-155 expression during heart failure in mice, indicating a role for non-cardiomyocyte-derived miR-155 in the immune pathogenesis of heart failure as well.Reference van de Vrie, Heymans and Schroen ⁶⁷ Low let-7i levels was also associated with poor clinical outcome.Reference Satoh, Minami, Takahashi, Tabuchi and Nakamura ⁶⁸

Very recently, new data have indicated that miR-22 acts as an integrator of Ca (+2) homeostasis and myofibrillar protein content during stress in the heart, and therefore shed light on the mechanisms that enhance propensity towards heart failure.Reference Gurha, Abreu-Goodger and Wang ⁶⁹ Alterations in miR expression have been observed during the process of right ventricular remodelling and in the gene regulatory pathways, leading to right ventricular hypertrophy and right ventricular failure.Reference Reddy, Zhao and Hu ⁷⁰ Interesting observations made in this study include differential regulation of miRs between the right and left ventricles. MiR-34a, miR-28, miR-148a, and miR-93 were upregulated in right ventricular hypertrophy/right ventricular failure, but remained downregulated or unchanged in left ventricular hypertrophy/left ventricular failure. Therefore, dysregulation of these miRs may contribute to the increased susceptibility of right ventricular hypertrophy to heart failure.Reference Reddy, Zhao and Hu ⁷⁰ MiR-21 regulates gene expression in multiple cell types in the heart. MiR-21 is profibrotic in fibroblasts, anti-apoptotic in cardiomyocytes, anti-angiogenic in endothelial cells, and anti-inflammatory in immune cells. Direct targets of miR-21 responsible for these effects include PDCD4 (phosphatase and tensin homologue, anti-inflammatory), SMAD7, Spry1 (sprouty homologue 1), PTEN, RhoB (ras homologue gene family member B), and FasL (fas ligand).Reference Tijsen, Pinto and Creemers ⁶⁰

Characterisation of miRs in heart failure may lead to new therapies including miRs/antagomiRs perhaps combined with gene therapy to treat heart failure. SERCA2a gene therapy for failing hearts was shown to restore miR-1 expression by a pathway involving Akt/FoxO3A that normalised the expression of the sodium–calcium exchanger-1 (NCX1) and improved cardiac function.Reference Kumarswamy, Lyon and Volkmann ⁷¹ These findings are supported by several studies that indicate that miR-1 plays a protective role against decompensated cardiac hypertrophy and heart failure.Reference Ikeda, He and Kong ^{72 –} Reference Care, Catalucci and Felicetti ⁷⁴ Although gene therapy for adult heart failure is still in the trial stage, whether miR-mediated therapy could be applied usefully to paediatric heart failure patients needs to be elaborated.

MiRs in myocarditis and cardiomyopathy

Myocarditis represents a serious cause of cardiac dysfunction in children,Reference Saji, Matsuura and Hasegawa ⁷⁵ which results in chronic dilated cardiomyopathy and death in up to 20% of the affected children.Reference Feldman and McNamara ⁷⁶ The pathogenesis of the disease is poorly understood, morbidity and mortality are high, and currently employed treatment strategies have little impact on improving the outcome. Only very recently, miRs were identified to be involved in viral myocarditis pathogenesis and susceptibility. It has been suggested that miRs possibly play a role in the pathogenesis of viral myocarditis through regulation of ion channel protein expression and adverse immune response to cardiotropic viruses.

MiR-1 may play a major role in myocarditis. MiR-1 is characteristically upregulated in viral myocarditis and causes suppression of Cx43,Reference Xu, Ding and Shen ⁷⁷ the main protein forming gap-junction channels in ventricular myocardium that allows electrical coupling and communication between adjacent cardiomyocytes,Reference Xu, Ding and Shen ⁷⁷ revealing the ability of a miR to regulate the expression of an ion channel protein in viral myocarditis. An antiviral activity for anti-miR-1 (AmiR-1) and AmiR-2 has been detected in Coxsackie virus B3 myocarditis.Reference Ye, Liu, Hemida and Yang ⁷⁸ The application of pRNA technology in the treatment of Coxsackie virus B3 infection and viral myocarditis in this study may be further developed as a system for RNAi-based drug design and delivery. The inflammatory miR-155 is also upregulated during acute myocarditis. It contributes to the adverse inflammatory response to viral infection of the heart and has been identified as a potential therapeutic target.Reference Corsten, Papageorgiou and Verhesen ⁷⁹

Hypertrophic cardiomyopathy and dilated cardiomyopathy

Hypertrophic cardiomyopathy and dilated cardiomyopathy constitute a group of primary myocardial disorders that are associated with miR dysregulation and lead to childhood death. Cardiomyopathies are typified by repeated re-hospitalisation and/or require cardiac transplantation within 1 year of the first admission.Reference Jansen, van Veen, de Bakker and van Rijen ⁸⁰ Metabolic or syndromic causes are identified in >35% of children with hypertrophic cardiomyopathy and dilated cardiomyopathy.Reference Kindel, Miller and Gupta ⁸¹

It is believed that miRs play key roles in maintaining cardiomyocyte integrity and that their dysregulation contributes to the pathogenesis of decompensated hypertrophic cardiomyopathy and progression to dilated cardiomyopathy.Reference Rao, Toyama and Chiang ⁸² Hypertrophic growth and myocyte disarray resulting in dilated cardiomyopathy and heart failure have been observed in mouse models with cardiac overexpression of miR-195Reference van Rooij, Sutherland and Liu ⁸³ and knockout mice for miR-133.Reference Liu, Bezprozvannaya and Williams ²² An additional study showed dysregulation of cardiac contractile proteins and profound sarcomeric disarray leading to rapidly progressive dilated cardiomyopathy in Dicer mutant mice.Reference Chen, Murchison and Tang ¹⁷

MiRs-142-3p and -5p are repressed by serum-derived growth factors in cultured cardiac myocytes and this may reflect similar changes in cardiac hypertrophy in vivo. Downregulation of miR-142 is a critical element of adaptive hypertrophy and mediates cytokine-induced survival signalling during cardiac growth in response to haemodynamic stress. Furthermore, miR-142 was found to be a global inhibitor of cytokine signalling and function in the myocardium, in part through its ability to target gp130 and downregulate the expression of α-Actinin.Reference Sharma, Liu, Wei, Yuan, Zhang and Bishopric ⁸⁴ miR-142 also represses multiple components of the Nuclear Factor-Kappa B pathway, acting as a critical regulator of immune response in myocardial tissue.Reference Sharma, Liu, Wei, Yuan, Zhang and Bishopric ⁸⁴

Dilated cardiomyopathy patients show decreased levels of let-7i, miR-126, and miR-155 in endo-myocardial tissues relative to controls.Reference Satoh, Minami, Takahashi, Tabuchi and Nakamura ⁶⁸ A decreased level of let-7i specifically has been associated with poor clinical outcome in patients with dilated cardiomyopathy. Similarly, miR-208 was found to be increased and shown to be a strong predictor of clinical outcomes for patients with dilated cardiomyopathy.Reference Satoh, Minami, Takahashi, Tabuchi and Nakamura ⁸⁵ A different panel of miRs is expressed in patients with hypertrophic cardiomyopathy.Reference Palacin, Reguero and Martin ⁸⁶ Patients with Friedreich ataxia cardiac hypertrophyReference Kelly, Bagnall and Peverill ⁸⁷ have polymorphism of the miR-155 binding sites in the angiotensin II receptor, type 1 gene promoter that may contribute to the hypertrophy phenotype.

Further identification of the post-transcriptional basis for myocarditis and associated cardiomyopathies through unravelling the roles of miRs, their regulation, and targets is urgently required to provide new treatment strategies and disease outcomes for these poorly understood conditions.

MiRs and arrhythmias

MiRs regulate all properties of cardiac excitability including conduction, repolarisation, automaticity, Ca²⁺ handling, spatial heterogeneity, apoptosis, and fibrosis. In addition to the wide range of actions on the myocardium, miRs are involved in the regulation of expression of a variety of proteins associated with the maintenance of the electrical properties of the heart.Reference Wang ⁸⁸ Symptomatic arrhythmias are responsible for 5% of all emergency hospital admissions in paediatrics. Although mostly benign in nature, arrhythmias can be life threatening. Childhood arrhythmias are unlikely to resolve spontaneously and may need long-term anti-arrhythmic treatment or catheter ablation.Reference Massin, Benatar and Rondia ⁸⁹

Atrial fibrillation is rare in children, but studies of the role of miRs have been quite extensive. MiR-1 levels are greatly decreased in atrial fibrillation patients, causing upregulation of Kir2.1 subunits with consequent shortening of the terminal phase of atrial electrical remodelling and sustained atrial fibrillation.Reference Girmatsion, Biliczki and Bonauer ⁹⁰ MiR-328 is increased in atrial fibrillation and contributes to adverse electrical remodelling, partially through targeting L-type Ca²⁺ channel genes.Reference Lu, Zhang and Wang ⁹¹ MiRs are also found to be differentially expressed in mitral stenosis patients with atrial fibrillation compared with those without atrial fibrillation. MiR-1202 was the most downregulated miR in these patients.Reference Xiao, Liang and Zhang ⁹² To our knowledge, no target or function of this miR has been reported. The prevalence of ventricular arrhythmia increases in children and adolescents with structural cardiac disease or cardiac surgery, putting them at significant risk for cardiac syncope and sudden cardiac death.Reference Serwer ⁹³ Alterations in cardiac miRs including miR-1, miR-133 and ion channel expression predispose patients to ventricular tachycardia. In patients with advanced non-ischaemic cardiomyopathy with ventricular tachycardia, both miRs-1 and miR-133a and their target mRNAs encoding ion channels were downregulated.Reference Amin, Giudicessi and Tijsen ⁹⁴ Consistent with this regulation, miR-1 overexpression was found to exacerbate arrhythmogenesis by direct repression of KCNJ2 and GJA1. GJA1 encodes connexin 43, the main cardiac gap-junction channel responsible for intercellular conductance in the ventricle.Reference Jongsma and Wilders ⁹⁵ KCNJ2 encodes the potassium inwardly rectifying channel.

MiR-1 enhances cardiac excitation–contraction coupling by selectively increasing phosphorylation of the L-type Ca²⁺ channels and ryanodine receptors (RyR2). It does this by disrupting the localisation of the protein phosphatase PP2A to these channels. Through translational inhibition of the PP2A regulatory subunit B56, miR-1 causes CaMKII-dependent hyperphosphorylation of RyR2, enhances RyR2 activity, and promotes arrhythmogenic sarcoplasmic reticulum Ca²⁺ release.Reference Jongsma and Wilders ⁹⁵ Muscle-specific miR-1 has also been identified as a cardiac arrhythmia-related miR in human and rat hearts after ischaemia. As discussed above, miR-1 targets the genes GJA1 and KCNJ2, thereby causing slowing conduction and depolarising the cytoplasmic membrane.Reference Lin, Gao, Alarcon, Ye and Yun ⁵¹ In normal or infarcted hearts, overexpression of miR-1 exacerbated arrhythmogenesis, whereas elimination of miR-1 by an antisense inhibitor in infarcted hearts relieved arrhythmogenesis.Reference Yang, Lin and Xiao ^{96 ,} Reference Terentyev, Belevych and Terentyeva ⁹⁷

MiRs as a paediatric cardiovascular biomarker and therapeutic targets

The identification of distinct circulating miRs may impact the development of specific miRs as biomarkers in paediatric cardiovascular diseases, especially for foetal congenital heart defects.Reference Yu, Han and Hu ⁹⁸ Placental-expressed miRs have been detected in maternal plasma and can be associated with congenital heart diseasesReference Kotlabova, Doucha and Hromadnikova ⁹⁹ and may be useful molecular markers for monitoring pregnancy-associated diseases. This discovery will open new possibilities for non-invasive and early prenatal diagnosis, allowing for early interventional and/or surgical treatment that are important to improve the prognosis of neonates with congenital heart diseases. A novel functional miR SNP rs11614913 in miR-196a2 was found to be a predictor of congenital heart diseases.Reference Xu, Hu and Xu ¹⁰⁰ Circulating miRs are also emerging as biomarkers in heart failure,Reference Tijsen, Creemers and Moerland ¹⁰¹ with increased plasma miR-1 being the front leader.Reference Cheng, Tan and Yang ¹⁰²

The identification of miRs that are dysregulated during the development of obesity could provide obesity biomarkers for early clinical diagnosis.Reference McGregor and Choi ¹⁰³ Several miRs are dysregulated in coronary artery disease patients and are detectable in peripheral blood mononuclear cells.Reference Hoekstra, van der Lans and Halvorsen ¹⁰⁴ These miRs are expressed in endothelial cells and show significantly reduced levels of miR-126, the miR-17/92 cluster (miR-17, miR-20a, and miR-92a), miR-130a, miR-221, miR-21, and members of the let-7 family.Reference Fichtlscherer, De Rosa and Fox ¹⁰⁵ This can help in early and non-invasive detection of asymptomatic coronary artery disease in obese children, especially those with other risk factors of metabolic syndrome, allowing for very early therapeutic interventions to slow or even reverse the atherosclerotic lesions.Reference Omran, Elimam, He, Peng and Yin ¹⁰⁶

MiRs are not only serving as potential biomarkers for early detection and diagnosis of disease, but also as therapeutic targets. Altering expression levels of disease-causing miRs by either their overexpression or inhibition are considered to be of tremendous therapeutic potential for the treatment of cardiovascular disease.Reference Fang, Song and Tian ¹⁰⁷ Antagonising endogenous miR-33 has been suggested as a therapeutic strategy for treating metabolic syndrome through regulation of fatty acid metabolism and insulin signalling.Reference Davalos, Goedeke and Smibert ¹⁰⁸ Observational and functional studies of miR-122 have highlighted it as a potential therapeutic target for the treatment of hypercholesterolaemia.Reference Esau, Davis and Murray ¹⁰⁹ Potentially, miR-27a mimics could be used to regulate pre-adipocyte proliferation and may evolve into useful anti-adipogenic drugs.Reference Rosen and MacDougald ¹¹⁰ MiR-1 and miR-133 target pacemaker channels such as HCN2 and HCN466, with miR-1 confirmed to have an arrhythmogenic actionReference Terentyev, Belevych and Terentyeva ⁹⁷ that could be reversed by knocking down miR-1 using its specific inhibitor antisense oligonucleotides.Reference Yang, Lin and Xiao ⁹⁶ The development and optimisation of AmiRs has great therapeutic potential. The safe, effective, and targeted delivery of in vivo RNA therapeutics remains an important challenge for clinical development. Another potential use of miRs to consider is their use for the diagnosis of individuals at risk for a specific disorder. Based on the observation that common single-nucleotide polymorphisms in pre-miRshas-miR-196a2 and hsa-miR-499 are associated with a significant increase in the risk of dilated cardiomyopathy,Reference Zhou, Rao and Peng ¹¹¹ suggests a possible benefit to use miRs for screening.

MiRs and cardiac stem cells

Cardiovascular regenerative medicine aims to restore damaged myocardium, both vasculature and muscle. Successful bone marrow stem cell therapy for myocardial regeneration in an infant with hypoplastic left heart syndrome has been reported.Reference Rupp, Zeiher and Dimmeler ¹¹² The regenerative capacity of human cardiac progenitor cells in young patients with non-ischaemic congenital heart defects showed that human cardiac progenitor cells are functional and also have potential in congenital cardiac repair.Reference Mishra, Vijayan and Colletti ¹¹³

MiRs have been shown to regulate multiple steps of stem cell growth, self-renewal, and differentiation including stem cell pluripotencyReference Mallanna and Rizzino ¹¹⁴ lineage specification,Reference Ivey, Muth and Arnold ¹² embryonic stem cell differentiation,Reference Gan, Schwengberg and Denecke ¹¹⁵ stem cell self-renewal,Reference Shekar, Naim, Sarathi and Kumar ¹¹⁶ regulating embryonic stem cells identity,Reference Laurent, Chen and Ulitsky ¹¹⁷ reprogramming of somatic cells to pluripotent cells,Reference Lakshmipathy, Davila and Hart ¹¹⁸ and allogeneic stem cell transplantation.Reference Wei, Wang and Qu ¹¹⁹ A human myocardial precursor derived from human cardiac progenitor cells that gives origin to atrial, ventricular, and specialised conduction cardiomyocytes has been recently identified.Reference Ritner, Wong and King ¹²⁰

MiRs have been identified to play roles in endothelial progenitor and cardiac myocyte specification and differentiation.Reference Small and Olson ^{121 ,} Reference Evans, Moretti and Laugwitz ¹²² MiR-99b, miR-181a, and miR-181b are considered to comprise an endothelial miR signature. These miRs play essential roles in the differentiation of pluripotent human cardiac progenitor cells to endothelial progenitor and endothelial cells.Reference Kane, Howard and Descamps ¹²³ MiR-125b is important during human embryonic stem cell differentiation into myocardial precursors and cardiomyocytes. MiR-125b also plays a regulatory role in the early stages of human embryonic stem cells differentiation, possibly by targeting Lin28, a miR-binding protein that binds to and enhances the translation of the insulin-like growth factor 2 mRNA. Lin28 has also been shown to bind the let-7 pre-miR and block production of mature let-7 in endothelial progenitor cells. MiR-125b appears to induce the formation of mesoderm, and cardiac mesoderm from human embryonic stem cells. Overexpression of miR-125b was shown to mediate upregulation of the early cardiac transcription factors GATA4 and Nkx2-5 and accelerate progression of human embryonic stem cell-derived myocardial precursors to an embryonic cardiomyocytes phenotype. These findings suggest that manipulation of miR-125b-mediated pathways could provide a novel approach to directing the differentiation of human embryonic stem cell-derived cardiomyocytes for cell therapy applications.Reference Wong, Ritner and Ramachandran ¹²⁴ These studies shed insights into the suitability of human embryonic stem cell-derived cardiomyocytes for therapies not only to heart attack victims, but also to those bearing the burden of other diseases where the child's heart is damaged or not functioning properly.

MiR-1 and miR-499 play differential roles in cardiac differentiation of human embryonic stem cells in a context-dependent manner; miR-499 promotes ventricular specification of human embryonic stem cells and miR-1 facilitates electrophysiological maturation.Reference Fu, Rushing and Lieu ¹²⁵ MiR-499 further promotes the differentiation of human cardiac stem cells into mechanically integrated cardiomyocytes, a function that offers great hope for the treatment of human heart failure.Reference Hosoda, Zheng and Cabral-da-Silva ¹²⁶ An miR pro-survival cocktail of miR-21, miR-24, and miR-221 is expected to improve the engraftment of transplanted cardiac progenitor cells and therapeutic efficacy for treatment of ischaemic heart disease through overcoming the low survival of the transplanted cell.Reference Hu, Huang and Nguyen ¹²⁷

It is noteworthy that miR-1 is involved in cardiac development, function, pathology, and treatment. The development of human embryonic stem cells lines and/or induced pluripotent stem cell with enhanced ability to differentiate into cardiomyocyte tissue holds great promise for several paediatric cardiovascular researches, especially if immunological rejection issues can be resolved.Reference Mishra, Vijayan and Colletti ¹¹³ The elucidation of the role of miRs in this area will aid in the development of new approaches for paediatric cardiovascular disease profiling and cell therapy.