The role of post‐translational modifications in cardiac hypertrophy

Abstract Pathological cardiac hypertrophy involves excessive protein synthesis, increased cardiac myocyte size and ultimately the development of heart failure. Thus, pathological cardiac hypertrophy is a major risk factor for many cardiovascular diseases and death in humans. Extensive research in the last decade has revealed that post‐translational modifications (PTMs), including phosphorylation, ubiquitination, SUMOylation, O‐GlcNAcylation, methylation and acetylation, play important roles in pathological cardiac hypertrophy pathways. These PTMs potently mediate myocardial hypertrophy responses via the interaction, stability, degradation, cellular translocation and activation of receptors, adaptors and signal transduction events. These changes occur in response to pathological hypertrophy stimuli. In this review, we summarize the roles of PTMs in regulating the development of pathological cardiac hypertrophy. Furthermore, PTMs are discussed as potential targets for treating or preventing cardiac hypertrophy.


| INTRODUC TI ON
The heart undergoes adaptive changes in response to long-term overload, namely myocardial hypertrophy. Physiological hypertrophy usually happens to pregnant women or athletes. 1 However, pathological cardiac hypertrophy is usually induced by stress stimulation or disease and is a typical pathological stage of diseases such as cardiomyopathy, myocardial infarction and diabetes. 2 Therefore, pathological cardiac hypertrophy is a predictor of many cardiovascular diseases and death in humans.
At the cellular level, the typical characteristics of pathological cardiac hypertrophy are increased cardiac muscle cell size, segregation of sarcomere structures, enhanced protein synthesis and foetal gene re-expression. 3 Cardiac hypertrophy is an adaptive response mediated by regulation at multiple levels, including the transcription, processing and translation of mRNAs and post-translational modifications (PTMs). 4 PTMs are more flexible and economical than regulation at the transcriptional level. PTMs usually regulate the activation/inactivation or degradation of pre-existing transcripts and proteins covalently modified by enzymes, resulting in rapid changes in the functions of pre-existing proteins, multiprotein complexes and subcellular structures in response to various physical and chemical stimuli. 5 Owing to PTMs, cardiomyocyte does not trigger de novo synthesis of proteins at the transcriptional level, which provides an approach to the saving of energy and material resources and compensates for the temporal-and-spatial weaknesses caused by transcriptional regulation. In addition, PTMs act as key regulators of proteins, contributing to changes in their diversity, localization, structure, interaction, roles etc, thus providing substantial complexity and elaborate regulation to the control of cardiac hypertrophy.
Therefore, comprehensive knowledge of PTMs involved in the development of myocardial hypertrophy will provide a better understanding of the molecular regulatory mechanism of pathological hypertrophy. This, in turn, will greatly benefit rational drug utilization and provide new treatment strategies for heart failure.

| PHOS PHORYL ATION
MAPKs, consisting of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 MAPKs, are well known to play important roles in mediating overload or pathological insult-induced cardiac hypertrophy. 6 For example, cardiomyocyte-specific expression of MEK-1 significantly induced ventricular concentric hypertrophy by phosphorylating ERK1/2 in the heart (Table 1). 7 ERK5 was also shown to play an essential role in the development of cardiac hypertrophy. 8 The Ca 2+ /calmodulin signalling pathway reportedly plays an important role in the occurrence of ventricular arrhythmias in hypertrophic cardiomyopathy and cardiac hypertrophy. 9 Ersilia et al showed that the CaMKII-ERK pathway was essential for developing cardiac hypertrophy and the impairment of their interaction provided a promising therapeutic modality to attenuate myocardial hypertrophy. 10 Recently, activation of ERK/glycogen synthase kinase-3(GSK3) induced by angiotensin II was shown to phosphorylate heat shock factor 1 (HSF1), resulting in degradation of RNF126, which promoted the expression of insulin-like growth factor II receptor (IGF-IIR) and ultimately induced myocardial hypertrophy (Table 1). 11 Thus, targeting HSF1 could be a promising strategy to prevent pathological cardiac hypertrophy.
Kojonazarov et al showed that inhibition of p38 MAPK activity improved heart function in response to pressure-loaded right ventricular hypertrophy by suppressing transcriptional pathways, including serum response factor and myocardin-related transcription factor A. 12 Regulator of G protein signalling 6 (RGS6) was reported to promote cardiac hypertrophy by activating apoptosis signal-regulating kinase1/p38 MAPK/JNK1/2 signalling. 13 A deficiency of JNKinteracting protein 3 could alleviate cardiac hypertrophy through inactivating the JNK pathway and might become a promising therapeutic target for treating cardiac hypertrophy and heart failure. 14 AKT, a serine/threonine kinase, is activated and phosphorylated by PDK1 and PDK2 at residues Thr308 and Ser473 respectively. 15 As a key molecule for cardiac hypertrophy, AKT activation can further phosphorylate many downstream proteins and thereby positively and negatively regulate diverse signalling pathways. AKT has been shown to promote cardiac hypertrophy through regulating several signalling pathways, such as PI3K/AKT/GSK3β, PI3K/AKT/mTOR and the FAK/AKT signalling. 16,17 Knockdown of protein kinase D (PKD) was shown to attenuate pressure overload-induced cardiac hypertrophy by promoting autophagy via AKT/mTOR pathway. 19 Dimethyl fumarate, a methyl ester of fumaric acid, is approved by the Food and Drug Administration for the treatment of relapsing/ remitting multiple sclerosis and psoriasis. Dimethyl fumarate was shown to protect against ISO-induced cardiac hypertrophy by decreasing the levels of p-ERK1/2 and increasing the level of p-AKT. 20 AMPK, a serine/threonine kinase, is activated and phosphorylated by LKB1 at residue Thr172 (Table 1). 21 36 Therefore, the exact regulatory network followed by protein kinase-mediated autophagy in cardiac hypertrophy needs further investigation in the future.
Together, the above-mentioned findings suggest that phosphorylation is essential for promoting or attenuating cardiac hypertrophy in various signal pathways.

| DUAL-S PECIFI CIT Y MAPK PH OS PH ATA S E S
A previous study has shown that DUSPs act as critical regulators of cardiac growth and remodelling by dynamically regulating the MAPK signalling pathway ( In brief, phosphorylation modifications play important roles in the regulation of cardiac hypertrophy and may prove to be promising targets for therapeutic development.

| UB IQUITINATION
Ubiquitination, a widely distributed PTM of proteins, regulates the timely functions of proteins. Recently, ubiquitin-proteasome system (UPS) proteins, E3 ligases and deubiquitylation enzymes (DUBs) were found to play important roles in the development of cardiac hypertrophy ( Figure 1; Table 3). 42  Cardiac fibrosis-induced pressure overload is an important step of maladaptive hypertrophy and ubiquitination of TRAF6 and RIP1, mediated by ligase E3 Pellino1, contributes to the activation of NF-κB and AP-1, resulting in increased expression of transforming growth factor-β1 in cardiac fibroblasts ( Figure 1). 55 In addition, pressure overload-induced cardiac maladaptive remodelling and dysfunction were mediated by deubiquitinating enzyme CYLD, which contributes to interrupt the ERK-and p38-/AP-1 and c-Myc pathways, resulting in suppressing expression of Nrf2 and Nrf2-operated antioxidative capacity. 56 Furthermore, deubiquitinating enzyme F I G U R E 1 Ubiquitination-mediated signalling pathways of cardiac hypertrophy. Ubiquitination plays an important role in cardiac hypertrophy by regulating the TAK1-JNK1/2/p38, NF-κB signalling, Ca 2+ /calmodulin, oxidation stress, ERK signalling pathways. In these pathways, pressure overload or other hypertrophic stimuli can induce E3 ligases or DUBs to activate MAPKs or other signalling pathways, ultimately regulating nuclear transcription factors to promote growth USP14 suppressed the progression of cardiac hypertrophy by increasing phosphorylation of glycogen synthase kinase-3β. 49 Recently, the E3 ubiquitin ligase, Muscle-specific RING finger protein-1 (MuRF1), was reported to mono-ubiquitinate thyroid hormone receptor α (TRα) to enhance its interaction with CAP350 and transcriptional activity in the nuclear compartment. 57 MuRF1 was also reported to attenuate pathological cardiac hypertrophy via promoting degradation of calcineurin A. 58 In addition, TCAP, which is down-regulated by the E3 ubiquitin ligase, MDM2, is involved in cardiac hypertrophy (Figure 1). 59

| SUMOYL ATION
The small ubiquitin-like modifier (SUMO) system catalyses classical ubiquitin-like post-translational protein modifications that are universally involved in cellular activities such as cell cycle regulation, genome stabilization, chromatin remodelling and transcription. 64 SUMOylation is also involved in cardiovascular diseases including cardiac hypertrophy. 65 For example, SUMO-1 is involved in heart failure by specifically mediating SUMOylation of SERCA2a (Table 4).
Interestingly, SUMO-1 is significantly reduced in mice and human patients with heart failure and heart failure was observed in mice following the deletion of cardiomyocyte-specific SUMO-1. As a result, the SUMOylation of cardiac SERCA2a was significantly decreased ( Figure 2 This increases the expression of IGF-IIR and induces hypertrophy (Table 4). 70 In addition, Wang et al reported that the overexpression of myofibrillogenesis regulator 1(MR-1) directly induced myocardial hypertrophy by enhancing the SUMOylation of myomesin-1 ( Figure 2). 71 In contrast to these findings, the activation of calcineurin/ nuclear factor of activated T cell (NFAT) signalling, and cardiomyocyte hypertrophy induced by SUMO2, are independent of

| ACE T YL ATION AND ME THYL ATION
Emerging evidence suggests that epigenetic modifications of histones, such as acetylation and methylation, are essential for the regulation of gene expression during the progression of cardiac hypertrophy. 89 The correct expression of genes in cardiomyocyte is the basis for normal cardiac function. Thus, abnormal gene expression may cause heart dysfunction. Papait et al found that histone methyltransferase G9a regulated key epigenetic changes during the progression of cardiac hypertrophy ( Figure 3). Hence, methylation was essential for cardiomyocyte homoeostasis and hypertrophy. 90 Likewise, the histone trimethyllysine demethylase, JMJD2A, promoted cardiac hypertrophy in response to hypertrophic stimulation in mice and induced an increase in the expression of hypertrophy markers including B-type natriuretic peptide and natriuretic peptide A in pluripotent stem cell-derived cardiomyocyte (Table 5). 91,92 The histone demethylase, PHF8, was also observed to attenuate cardiac hypertrophy upon cardiac overload 93 (Figure 3).
In the following section, we focus on the roles of acetylation in the development of cardiac hypertrophy progression (Figure 3).
Previous studies reported the key function of histone deacetylases (HDACs) in the regulation of pathological heart growth. Class II HDACs maintain normal cardiac function and size by mediating the expression of MEF2 transcription factors and other factors. 94 Recent studies reported that Class II HDACs were essential for vascular smooth muscle cell hypertrophy and hyperplasia through the CaMKIIα/protein kinase D1/HDAC4/GATA6 pathway. 56 In addition, cardiomyocyte hypertrophy was attenuated by transcription factor 3 (ATF3), binding with the Map2K3 promoter, resulting in recruiting HDAC1 and suppressing MAP2K3-p38 Signalling. 95 Furthermore, the class III HDAC, sirtuin 1 (SITR1), reportedly prevented cardiomyocyte hypertrophy by negatively regulating the acetylation and phosphorylation levels of protein kinase C-ζ (Table 5). 96 108 Recently, hypertension-induced cardiac hypertrophy was reported to be protected by sirtuin 3 (SIRT3), deacetylating Pink1/Parkin, resulting in mitophagy and reduction of ROS production. 109 Notably, sirtuin 6 (SIRT6) regulated the progression of cardiac hypertrophy by deacetylating H3K9 to inhibit IGF-Akt signalling pathway (Table 5). 110 SIRT6 also reported to prevent cardiomyocyte hypertrophy by inhibiting the expression of transcription 3 (STAT3). 111 Finally, in SIRT6-deficient hearts, SIRT1 was observed to be deacetylated and activated Akt signalling pathways. 112 In conclusion, these findings highlight the critical role of both methylation and acetylation in the initiation, progression and outcome of maladaptive cardiac remodelling and dysfunction and HDAC inhibitors are promising drugs to target cardiac hypertrophic signalling for heart failure treatment.

| THE MULTIFACE TED CONTROL OF P TM
It is well recognized that cardiac hypertrophy is mediated at several levels, including gene transcription, processing and translation of mRNAs and PTMs. PTMs act as key regulators of proteins, occurring as a modification at a single residue or combining effects over multiple sites undergoing the same or different modifications. 113 Cells need to be connected to various PTM signals and coordinated with each other to properly regulate cardiac hypertrophy. Furthermore, emerging evidence has highlighted important roles for crosstalk between different pairs of PTMs, such as ubiquitylation-phosphorylation, 21 SUMOylation-phosphorylation, 65 acetylation-phosphorylation, 114 O-linked glycosylation-phosphorylation, 80 and acetylation-methylation. 63 Figure 1). SUMO-1 is involved in heart failure by specifically mediating SUMOylation of SERCA2a. However, phosphorylation of SERCA2a is essential for SUMOylation of SERCA2a mediated by SUMO-1 in mice and human patients with heart failure (Figure 2). 65 AMPK is a hetero-trimeric complex, which is activated by phosphorylation on the residue Thr172. 115 In addition, AMPK inhibits O-GlcNAcylation by mainly regulating phosphorylation of GFAT and AMPK activation counteracts cardiac hypertrophy by reducing O-GlcNAcylation of proteins such as troponin T. 80 Acetylation and trimethylation on H3K27 play opposing roles at the promoter regions of genes involved in cardiac hypertrophy. 116 A previous study has shown that SIRT1 attenuated the PKC-ζ activity via mediating the interplay of acetylation and phosphorylation in cardiac hypertrophy 96 (Figure 3). In conclusion, these findings suggest that crosstalk between different pairs of PTMs is essential for cardiac function.
Future work in this field is needed to determine the global mechanistic actions of these PTMs in the heart.

| CON CLUS I ON S AND PER S PEC TIVE S
A considerable number of studies have shown that myocardial hypertrophy is a phenomenon in which cardiac cells transform from a mature 'contractile state' to an 'embryonic synthesis state' and is the primary pathophysiological process in the development of heart failure. 117 Myocardial hypertrophy can lead to reduced blood pressure, cardiac cell hypertrophy and apoptosis, decreased ventricular compliance and impaired ejection function, resulting in a vicious cycle of worsening cardiac functions. Overall, myocardial hypertrophy has become an increasingly important factor in the field of cardiovascular disease. Therefore, it is particularly important to explore its mechanism.
As reported in the studies reviewed in this article, myocardial

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.