LncRNAs in cardiac hypertrophy: From basic science to clinical application

Abstract Cardiac hypertrophy is a typical pathological phenotype of cardiomyopathy and a result from pathological remodelling of cardiomyocytes in humans. At present, emerging evidence demonstrated the roles of long non‐coding RNAs (lncRNAs) in regulating the pathophysiological process of cardiac hypertrophy. Herein, we would like to review the recent researches on this issue and try to analysis the potential therapeutic targets on lncRNA sites. Studies have revealed both genetic mutations related hypertrophic cardiomyopathy and the compensative cardiac hypertrophy due to pressure overload, inflammation, endocrine issues and other external stimulations, share a common molecular mechanism of ventricular hypertrophy. The emerging evidence identified the abnormal expression of lncRNAs would leading to the impairment the function of sarcomere, intracellular calcium handling and mitochondrial metabolisms. Several researches proved the therapeutic role of lncRNAs in preventing or reversing cardiac hypertrophy. With the development of delivery system for small pieces of oligonucleotide, clinicians could design gene therapy approaches to terminate the process of cardiac hypertrophy to provide better prognosis.


| INTRODUC TI ON
Cardiac hypertrophy is considered as a pathological phenotype of hypertrophic cardiomyopathy (HCM) or a result from cardiomyocytes' remodelling due to a series of external stimulations. HCM, usually inherited in an autosomal dominant pattern with variable expressivity with age-and sex-related, is defined by the presence of left ventricular hypertrophy (LVH) which could not be clarified by abnormal hemodynamics overloading. 1,2 HCM has a heterogeneous clinical profile, ranging from asymptomatic personnel to symptomatic patients with severe hypertrophic remodelling, inducing diastolic dysfunction and ventricular arrhythmias which are represented as the two dominant features of HCM, leading the main cause of sudden cardiac arrest in young individuals. 3 As 1980s first discovered the mutation of MYH7 coding the protein of β-MHC associated with HCM, more than 2000 gene mutations related to 11 kinds of sarcomere proteins have been found to be related to HCM over the past 30 years. 4 With more and more gene mutations been verified, while around 50% phenotype positive patients still with genotype negative, lead difficult to treat patients directly via the gene mutation of individuals. Therefore, focusing on the downstream mechanisms to find a common target is becoming more and more urgency.
Besides the aetiology of genetic mutation for some specific HCM, the pathophysiological process of cardiac hypertrophy shares a common molecular mechanism of ventricular hypertrophy, which is associated with the sustain cardiac output, and chronically sustained stimulation of undesirable factors would deteriorate the pumping capacity of cardiac, causing multifactorial clinical syndrome, even heart failure. These compensatory and deteriorate changes in cardiomyocyte are associated with re-expression of foetal genes, which are mainly related to sarcomere contraction, ion signal regulation and activity, and mitochondrial function. [5][6][7] Increased myofilament Ca 2+ sensitivity has been identified in animal model and patients with pathological hypertrophy, leading changes of intracellular Ca 2+ homoeostasis and causing Ca 2+ -triggered arrhythmias, which could increase force development and adenosine triphosphate (ATP) consumption. 8,9 Mitochondrial disorganization and dysfunction in cardiac hypertrophy could cause energy supply and demands imbalance. 10 11 Meanwhile, lncRNAs could regulate cardiac pathological processes through multiple molecular mechanisms in cis and in trans, which are transcribed RNA molecules >200 nucleotides in length but have no potential for protein coding. 12,13 More and more studies found that lncRNAs might act an important role to regulate the intracellular calcium handling, metabolic state in various cardiovascular diseases, including HCM (Table 1). 14,15 In this review, we mainly present an overview to clarify the role of lncRNAs in the pathological process of cardiac hypertrophy further to benefit the work on targeting therapy.

| MOLECUL AR BA S IS OF C ARDIOMYOC Y TE HYPERTROPHY A SSO CIATED WITH LN CRNA S
Cardiac performance is relied on Ca 2+ and ATP, to pump blood to meet the energy demands of peripheral tissues, which depends filaments of sarcomere sliding. Sarcomere is the basic functional unit of the cardiomyocyte, made up by the thin and thick filaments containing a set of proteins. 16 The thin filament is mainly consisting of α-actin, troponin complex (Tn) containing TnT, TnC, TnI and tropomyosin. The thick filament is mainly composed of myosin heavy Key points 1. Several lncRNAs participate in the regulation of pathophysiological process in cardiac hypertrophy, interacting with the genes involving sarcomere, mitochondria and intracellular calcium transition.
2. Current evidence demonstrates the lncRNAs could be a promising target to attenuate the process of cardiac hypertrophy or even reverse the pathological phenotype.   Figure 1). 17,18 TnC could be activated by calcium-induced calcium release (CICR), inducing a conformational change in the Tn complex, resulting tropomyosin out of its groove on the actin filament, leading actin filament exposed. Myosin heads also named cross-bridges, with the binding domain of ATP, can interact with the exposed actin, along with ATP hydrolysing to adenosine diphosphate (ADP) and inorganic phosphate (Pi), which could provide the energy to pull the thin filament towards the centre of the sarcomere. Many sarcomeres contracting in series contract the myofibrils, causing shortening of the whole cardiac fibre ( Figure 1). 19,20 Besides, the Ca 2+ is critical to generate contraction as myofilament activation and force development is largely dependent on intracellular Ca 2+ .The degree of myofilament Ca 2+ sensitivity is dynamically regulated by the Ca 2+ binding affinity of Tn complex. 21 TnT mutations account for approximately 15% of genotype-positive HCM cases. This subset of HCM cases are characterized by relatively mild or subclinical hypertrophy with a high incidence of sudden death, which may be associated with increased myofilament Ca 2+ sensitivity in the mutation. 22 Which also inspired us that the Ca 2+ binding affinity of Tn complex may be an integration point to understanding the gene mutation and clinical manifestations.
As mentioned above, the sarcomere contractile cycle is pow-

| LncRNAs regulate the homoeostasis of mitochondria
Mitochondrial is one of the most important organelles in cardiomyocytes. Nearly, all the biological activity is relied on the homoeostasis and normal function of mitochondria. Mitochondria is directly correlated between cardiac workload and oxygen consumption, which occupy ~30% of cardiomyocyte and generate more than 95% of ATP consumed by the heart. 27,28 Cardiac contraction is powered by ATP administration, which is produced within mitochondria through oxidative phosphorylation process on fatty acid, while glucose oxidation or glycolysis could also supply some energy under hypoxia condition. 27 Mitochondria is the multifunctional cell organelle involved in essential functions of cardiomyocytes, including maintain morphology of cardiomyocyte via mitochondrial dynamics process, reactive oxygen species (ROS) accumulation, calcium transportation by uniporter located on mitochondrial membrane, apart from energy generation. 29 Pathological hypertrophy is associated with mitochondria dysfunction. 27 The emerging evidence suggested that lncRNAs contribute to the synchronization of cellular biological pathways in HCM, via regulating the homoeostasis of mitochondria. So that, a series of non-coding RNAs, such as lncRNAs and miRNAs, have been identified to be involved in the mitochondrial dynamics controls, which may be a potential therapeutic to intervene the cardiac diseases. However, according to common sense, the interactions between enrolled lncRNAs and miRNAs need to be further demonstrated.
Beyond the morphology relationship between mitochondria and cardiomocyte, the metabolic alternations are another character during cardiac hypertrophy. Metabolic remodelling in pathological hypertrophy with shift from predominant long-chain fatty acid utilization to glucose utilization will result in an shortage in ATP consumption. 34 Carnitine palmitoyl transferase I (CPT1) need to be recruited in the first step in the transportation of fatty acids into mitochondria, which is encoded by CPT1b gene in heart and muscle cells. The activity of CPT1b is considered to guiding fatty acid oxidation. 35 While, lnc-Uc.323 has been documented it could regulate F I G U R E 2 The schematic diagram of the lncRNA and it is targeted protein in cardiomyocytes the expression of CPT1b via interacting with its coactivator of zeste homolog 2 (EZH2), which bound to the promoter of CPT1b via H3K27me3 to induce CPT1b down-regulation, protecting cardiomyocytes against hypertrophy (Figure 2). 36 The lnc-Uc.233 depletion mice presented a severe hypertrophic heart, providing evidence on the association between lncRNAs and metabolic disorder in regulation of cardiac hypertrophy.

| LncRNAs controls the function of calcium channels
Arrhythmia is always combined with HCM, which may be as- binding in the promoter of CaMKII in cardiomyocytes (Figure 2). 46 Lnc-H19 as the precursor of miR-675, which inhibit the expression of CaMKIIδ, is a negative regulator of cardiomyocyte hypertrophy ( Figure 2). 47 Calcineurin is serine/threonine protein phosphatase regulated by CaMKII, which plays an important role in the regulation of pathological hypertrophy. 48 Calcineurin could reciprocal repress with miR-133 to regulate cardiac hypertrophy. 49

| LN CRNA : A P OTENTIAL TARG E T FOR C ARDIAC HYPERTROPHY
The current pharmacologic therapy remains alleviating the symptoms of HCM patients rather than focusing on the underlying cause, such as using β-adrenergic receptor blocker to blunt the heart rate and increase diastolic filling time. With more and more genetic anal-

| VALIDATI ON OF A LN CRNA-BA S ED G ENE THER APY ON NON -VIR AL AND VIR AL CON CEP TIONS
Several approaches have been applied to generate the silence and overexpression oligonucleotide for lncRNAs. Simply, the lncRNA siRNA has been used to inhibit the site targeting for lncRNA. Besides, the antisense oligonucleotide (ASO) has been widely introduced to impair the function of lncRNAs. Beyond cardiovascular system, the application of ASO for lncRNA has been confirmed a promising therapeutic method for osteoarthritis, hepatology disease and cancer. While the lncRNA minics has been used to expression specific oligonucleotide (Table 1).
Moreover, some gene-editing method has also been taken into knock The Figure 3 demonstrated the common protocol to validate the ln-cRNA-based gene therapy to clinical applications ( Figure 3).

F I G U R E 3
The common protocol to validate the lncRNA-based gene therapy to clinical applications. A, The design strategies for the purpose of targeting lncRNAs. B, The non-viral and viral methods to establish a delivery system for lncRNA-based gene therapy. C, The validation program of lncRNAbased gene therapy from cell lines and cardiomyocytes' tissue verification, small and big animals' validation and finally launching a clinical trail

| CON CLUS ION
Over the past several decades, the molecular mechanisms on cardiac hypertrophy have made a further progression. However, there are still some big dilemmas to fully elucidating the pathological mechanisms for better treatment of cardiac hypertrophy. More and more evidence illustrated that lncRNAs, such as Mhrt, Plscr4 and H19, could act as an important participant in the complicated network in regulating the pathological process of hypertrophy, which involves in the functional homoeostasis among sarcomere, mitochondria and intracellular calcium transition, making them as a promising target for treatment of cardiac hypertrophy. This review demonstrated the function of lncRNAs in the processes of pathology hypertrophy, aimed to provide a new insight to explore the pathological mechanisms and to find novel therapeutic targets for disease modulation and prevention in cardiac hypertrophy.
Besides, the review presented current strategies to provide gene therapy and the program to validate a lncRNA-based therapeutic approach from bench site to bedside.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science Foundation of China (No. 81700360).

CO N FLI C T O F I NTE R E S T
The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.