MicroRNAs and Cardiovascular Disease

This first chapter of the book aims to provide readers the basic information necessary for better understanding the contents of other chapters. The chapter begins with the stories or history of discovery of miRNAs. From this section, readers can get a sense on how some groundbreaking scientific findings could be achieved from small but mysterious things. In the second section, the whole pathway of biogenesis of miRNAs is described in detail, from transcription to maturation. This is followed by the section on action of miRNAs describing how mature miRNAs are hooked up to the protein complex and guide the complex to target genes to execute repression of gene expression at the post-transcriptional level. Finally, some important issues related to miRNA function are introduced in the section entitled “Some conceptual notes about miRNA function.”

To date, more than 5000 miRNAs have been identified in animals across the lowest and the highest species, some 800 of them are found in humans. To have better understanding of how miRNAs function in the heart, it is necessary to have an idea about which miRNAs exist in the heart. This chapter focuses on cardiovascular-expressed miRNAs or the miRNA profiles in heart and vessel. Specifically, the miRNAs existing in the heart will be introduced by categories: cardiac-specific, muscle-specific, cardiac-enriched, and other cardiac-expressed, in detail on their genomic locations and relative expression levels in normal cardiac tissues. Alterations of miRNA signature under various diseased states of the heart are succinctly described and the precautions in interpreting the profiling data are stated

This chapter aims to summarize the available data on regulation of cardiac development and stem cell differentiation by miRNAs. Heart malformations occur in as high as 1% of newborns, presenting a significant clinical problem in our modern world. The first functional organ in the embryo is the heart and cardiovascular system and the heart is susceptible to congenital defects more than any other organ. Both intrinsic and extrinsic factors determine the development of the cardiovascular system. miRNA was initially described as being fundamental for developmental biology first in nematode worms and then in phylogenically more advanced organisms. Many defects of the miRNA machinery are incompatible with correct and/or continued development. On the other hand, pluripotency and cellular differentiation are intricate biological processes that are coordinately regulated by a complex set of factors and epigenetic regulators. As in other tissues, a distinct set of miRNAs is specifically expressed in pluripotent embryonic stem cells. This chapter describes the involvement of miRNAs in normal cardiac development, in congenital heart disease and Down syndrome, and in determining stem cell fate. In particular, the roles of miR-1, miR-133, miR-130a and miR-138 in cardiac development are described as these miRNAs have been experimentally studied in detail.

The aim of this chapter is to introduce the role of miRNAs in the pathological process of cardiac hypertrophy/heart failure. Cardiovascular disease is among the main causes of morbidity and mortality in developed countries. In response to stress, the adult heart undergoes remodelling process and hypertrophic growth to adapt to altered workloads and to compensate for the impaired cardiac function. Pathological hypertrophy results in loss of cardiac function and is the major predictor of heart failure and sudden death. Recent studies have established the role of miRNAs in cardiac hypertrophy/heart failure as causal factors or important regulators. miRNAs are aberrantly expressed in various animal models and in patients with heart failure. The miRNAs involved in cardiac hypertrophy/heart failure can in general be divided into two categories: anti-hypertrophic and pro-hypertrophic miRNAs. This chapter introduces the roles of miRNAs in experimental and clinical cardiac hypertrophy/heart failure. Detailed description is given of the well-studied pro-hypertrophic miRNAs miR-195, miR-208 and miR-23a, and anti-hypertrophic miRNAs miR-1, miR-133 and miR-9.

The aim of this chapter is to provide an overview on the role of miRNAs in myocardial ischemia, ischemia/reperfusion injury and ischemic preconditioning. Myocardial ischemia due to occlusion of coronary arteries constitutes the major cause of mortality and morbidity of humans worldwide by causing an array of injuries. Timely myocardial reperfusion remains the most effective treatment strategy for reducing myocardial infarct size, preventing left ventricular remodelling, preserving left ventricular systolic function and improving clinical outcomes. However, the full benefits of myocardial reperfusion are not realized, given that the actual process of reperfusing ischemic myocardium can independently induce myocardial injury. On the other hand, heart has endogenous cardioprotective capability against myocardial/reperfusion injury, called ischemic preconditioning. Recent studies indicate that miRNAs are implicated in all these different aspects of myocardial ischemia. This chapter describes the role of miR-1 and mR-133 in myocardial ischemia, miR-21, miR-29 and miR-320 in ischemia/reperfusion injury, and miR-21 and miR-199a in preconditioning.

Cardiomyocytes are excitable cells that can generate and propagate excitations; excitability is a fundamental characteristic of these cells, which is reflected by action potential, the changes of transmembrane potential as a function of time, orchestrated by ion channels, transporters, and cellular proteins. The electrical excitation evoked in muscles must be transformed into mechanical contraction through the so-called excitationcontraction coupling mechanism, and the proper contraction of cardiac muscles then drives pumping of blood to the body circulation. Arrhythmias are electrical disturbances that can result in irregular heart beating with consequent insufficient pumping of blood. Arrhythmias are often lethal, constituting a major cause for cardiac death, particularly sudden cardiac death, in myocardial infarction and heart failure. Recent studies have led to the discovery of microRNAs (miRNAs) as a new player in the cardiac excitability by fine-tuning expression of ion channels, transporters, and cellular proteins, which determines the arrhythmogenicity in many conditions. This review article will give a comprehensive summary on the data available in the literature. The basics of cardiac excitability are first introduced, followed by a brief introduction to the basics of miRNAs. Then, studies on regulation of cardiac excitability by miRNAs are described and analyzed.

Several processes including endothelial angiogenesis, vascular neointimal lesion formation, vascular inflammation process, lipoprotein metabolism, and hypertension are critically involved in antherogenesis or atherosclerosis. This chapter aims to introduce the role of miRNAs in endothelial angiogenesis. Angiogenesis is the growth of new blood vessels from pre-existing vessels. Functional endothelial cells are required for angiogenesis, a process involving proliferation, migration, and maturation of endothelial cells. Several miRNAs have been shown to critically regulate vascular angiogenesis and they can be classified into pro-angiogenic miRNAs and anti-angiogenic miRNAs. Pro-angiogenic miRNAs indentified to date include miR-130a, miR- 17~92 cluster, miR-210, miR-378, miR-7f, miR-27b, and miR-126. The known anti-angiogenic miRNAs include miR-320, miR-221 and miR-222. In addition, the miRNAs that are reportedly involved in tumor vascular biology are also introduced in this chapter.

Several processes including endothelial angiogenesis, vascular neointimal lesion formation, vascular inflammation process, lipoprotein metabolism, and hypertension are critically involved in atherosclerosis. This chapter aims to introduce the role of miRNAs in neointimal formation. Neointimal formation is a common pathological lesion in diverse cardiovascular diseases occurring at sites of subclinical atherosclerosis but are also classical hallmarks of restenosis after stenting, angioplasty, endarterectomy, and arterial transplantation. Neointimal growth is the balance between proliferation and apoptosis of vascular smooth muscle cells. A number of miRNAs, miR-21, miR-143, miR-145, miR-221, and miR-222, have been demonstrated to play important role in neointimal formation. Their corresponding target genes have also been established.

Several processes including endothelial angiogenesis, vascular neointimal lesion formation, vascular inflammation process, lipoprotein metabolism, and hypertension are critically involved in atherosclerosis. This chapter aims to introduce the role of miRNAs in vascular inflammation process. Atherosclerosis is now widely accepted to be an inflammatory disease, characterized by degenerative as well as proliferative changes and extracellular accumulation of lipid and cholesterol, in which an ongoing inflammatory reaction plays an important role in both initiation and progression/destabilization, converting a chronic process into an acute disorder. In early atheromotous plaques, inflammatory macrophages strive to alleviate the subendothelial accumulation of modified lipoproteins carrying cholesterol esters. Consequently, the further recruitment and migration of cells induce chronic inflammation. To date, only a few miRNAs have been shown to be involved in the vascular inflammation process. These include miR-126 in regulating adhesion molecules, and miR-155 and miR-125a in regulating inflammatory cytokine.

Several processes including endothelial angiogenesis, vascular neointimal lesion formation, vascular inflammation process, lipoprotein metabolism, and hypertension are critically involved in atherosclerosis. This chapter aims to introduce the role of miRNAs in lipoprotein metabolism. The low-density lipoprotein (LDL) when oxidized by oxygen free radicals and coming into contact with arterial wall, causes atherosclerotic lesions leading to increase in endothelial permeability and adhesiveness. In response to the damage to the artery wall and endothelial dysfunction, the immune system responds by recruiting white blood cells to adsorb the oxidized-LDL. The stimulation of lipid uptake into these cells by oxidized-LDL is critical to the initiation and development of atherosclerosis. miR-122 has been documented to regulate cholesterol synthesis and miR-125a to regulate lipid uptake in monocytes/macrophages.

Several processes including endothelial angiogenesis, vascular neointimal lesion formation, vascular inflammation process, lipoprotein metabolism, and hypertension are critically involved in atherosclerosis. This chapter aims to introduce the role of miRNAs in hypertension. Angiotensin II, the major bioactive peptide of the renin–angiotensin system, plays a crucial role in controlling various cardiovascular diseases, especially hypertension. Endothelium-dependent nitric oxide (NO) formation has been reproducibly demonstrated to be reduced in patients with essential hypertension compared with normotensive control subjects. Formation of NO in endothelial cells depends on an adequate and continuing supply of its key substrate, L-arginine. Studies on miRNAs in hypertension have been rather sparse, though miRNAs have been reported to play a role in hypertension. In particular, miR-155 regulates angiotensin II type 1 receptor and miR-122 regulates L-arginine transport.

The goal of this chapter is to discuss the regulation of cardiac fibrosis by miRNAs. Cardiac myocytes are normally surrounded by a fine network of collagen fibers. In the normal heart, two thirds of the cell population is composed of nonmuscle cells, the majority of which are fibroblasts. Cardiac fibrosis is the result of both an increase in fibroblast proliferation and extracellular matrix (ECM) deposition. A growing body of evidence indicates that, along with cardiomyocytes hypertrophy, diffusion of interstitial fibrosis is a key pathologic feature of myocardial remodelling in a number of cardiac diseases of different (e.g. ischemic, hypertensive, valvular, genetic, and metabolic) origin. The extracellular matrix (ECM) is a dynamic microenvironment; changes within ECM constitute the second important myocardial adaptation that occurs during cardiac remodelling. A subset of miRNAs is enriched in cardiac fibroblasts compared to cardiomyocytes. A number of studies have demonstrated the involvement of miRNAs in regulating myocardial fibrosis in the settings of myocardial ischemia or mechanical overload. Some miRNAs (miR-208 and miR-21) have been shown to favor fibrogenesis, being profibrotic miRNAs. Others including miR-29, miR-133, miR-30c, and miR-590 have been demonstrated to produce inhibitory effects on fibrogenesis, being anti-fibrotic miRNAs.

This chapter aims to discuss the regulation of cardiomyocyte apoptosis by miRNAs. Apoptosis is an active process that leads to cell death. Unlike necrosis, apoptosis is a complex endogenous gene-controlled event that requires an exogenous signal–stimulated or inhibited by a variety of regulatory factors, such as formation of oxygen free radicals, ischemia, hypoxia, reduced intracellular K+ concentration, and generation of nitric oxide. Apoptosis has been implicated in a variety of human disease including heart disease, Alzheimer’s disease, cancer, etc. To date, no less than 30 individual miRNAs are known to regulate apoptosis. The number in the list is expected to expand quickly with more studies. A number of miRNAs including miR-1, miR-29, and miR-320 are considered proapoptotic miRNAs. The miRNAs identified to date possessing antiapoptotic action include miR- 133, miR-21 and miR-199a. In addition, miR-21 has also been shown to produce proproliferative and antiapoptotic effects in vascular smooth muscle cells. This chapter provides detailed description of these individual miRNAs for their role in cardiomyocyte apoptosis

This chapter aims to introduce the role of miRNAs in regulating cardiac contraction. Cardiac contraction is triggered by excitation–contraction coupling: the cascade of biological events that begins with cardiac action potential and ends with myocyte contraction and relaxation. Cardiac muscle contraction is determined by the intrinsic contractile proteins: α- and β-myosin heavy chain (αMHC and βMHC). αMHC and βMHC are encoded by MYH6 and MYH7 genes, respectively, and their expression is species specific and varies in response to developmental and pathophysiological signaling alterations. Remarkably, studies revealed that myosin genes not only encode the major contractile proteins of muscle, but also act more broadly to control muscle gene expression and performance through a network of intronic miRNAs: the transcripts from these genes all contain pre-miRNAs. On the other hand, the cytoskeleton of cardiac myocytes consists of actin, the intermediate filament desmin, the sarcomeric protein titin, and α- and β-tubulin, which form the microtubules by polymerization. The loss of integrity of the cytoskeleton, with a resultant loss of linkage of the sarcomere to the sarcolemma and extracellular matrix, would be expected to lead to contractile dysfunction. miRNAs have been found to regulate both the contractile proteins (miR-208 and miR-21) and cytoskeleton proteins (miR-1 and miR- 133) to regulate cardiac contraction.

This chapter aims to introduce the role of miRNAs in regulating neurohormonal activation. The natural progression of heart failure is accompanied by the compensatory activation of cardiac and extracardiac neurohormonal systems and changes in the anatomy and function of the left ventricle. An array of biologically active molecules belong to the sympathetic adrenergic nervous system (norepinephrine) and renin–angiotensin– aldosterone system (RAS) (Ang II and aldosterone), which are responsible for maintaining cardiac output through increased retention of salt and water, peripheral arterial vasoconstriction, and contractility, as well as inflammatory mediators that are responsible for cardiac repair and remodeling. Although, the role of miRNAs in regulating the components of RAS and the adrenergic system is still poorly not well understood, several recent observations are worth noting. In particular, miR-155 is implicated in suppressing the levels of the Ang II type 1 receptor, and miR-21 can increase aldosterone secretion in human adrenal cells. This chapter describes very limited information in this regard.

This chapter aims to provide an implicit introduction to the role of miRNAs in regulating cardiac metabolism. The homeostasis of glucose, lipid, protein, and energy, which is critical for normal cardiovascular function, is maintained by cellular metabolism. Metabolic perturbation occurs in various types of cardiac disease, including myocardial ischemia, cardiac hypertrophy, heart failure, diabetic cardiomyopathy, atherosclerosis, etc. The depletion of high-energy-phosphate metabolites may contribute to heart failure, and a decreased PCr/ATP ratio has been found in cardiac muscle of heart failure patients and animal models of heart failure. A major determinant of glycolytic flux is glucose transport; glucose enters cardiac cells via the facilitative glucose transporters GLUT1 and GLUT4. Several miRNAs have been demonstrated to produce regulatory effects on GLUT4, cellular ATP level, and the pleiotropic factor IGF-1. A succinct summary on these studies is given in this chapter.

This chapter aims to discuss recent advances of circulating miRNAs as new and promising biomarkers for cardiac disease. The elucidation of miRomes between diseased and normal cardiovascular tissues or between different cardiovascular disease types, stages and grades, gives the chance to identify the miRNAs most probably involved in cardiovascular disease and to establish new diagnostic and prognostic markers. Recent findings suggest that circulating miRNAs may be plasma biomarkers for the diagnosis of lung, colorectal, and prostate cancers. These findings have been also tested for cardiovascular disease. miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. The levels of miRNAs in serum are reproducible and consistent among individuals of the same species. In particular, blood miR-1, miR-133, miR- 208a and miR-499 have been suggested as biomarkers of acute myocardial infarction; miR-208, miR-423-5p and some other miRNAs in the circulation are correlated with heart failure; and miR-122, miR-124 and miR-133 may be used to predict cerebral artery occlusion stroke.

miRNA interference (miRNAi) is a new concept that I proposed for the development of strategies and technologies to manipulate the function, stability, biogenesis, or expression of miRNAs and as such it indirectly interferes with expression of protein-coding mRNAs. This chapter aims to give an implicitly detailed introduction to some of the miRNAi strategies and technologies developed so far. The fundamental mechanism of miRNA regulation of gene expression is miRNA:mRNA interaction or binding. A key to interfere with miRNA actions is to disrupt the miRNA:mRNA interaction. In order to achieve this aim, one can either manipulate miRNAs or mRNAs to alter the miRNA:mRNA interaction. For miRNAs, one can either mimic miRNA actions to enhance the miRNA:mRNA interaction or to inhibit miRNAs to break the miRNA:mRNA interaction. Both gain-offunction and loss-of-function technologies are necessary tools for understanding miRNAs. The miRNAi technologies have opened up new opportunities for creative and rational designs of a variety of combinations integrating varying nucleotide fragments for various purposes and provided exquisite tools for functional analysis related to identification and characterization of targets of miRNAs and their functions in gene controlling program. In addition, the miRNAi technologies also offer the strategies and tools for designing new agents for gene therapy of human disease.