Original article
CYLD exaggerates pressure overload-induced cardiomyopathy via suppressing autolysosome efflux in cardiomyocytes

https://doi.org/10.1016/j.yjmcc.2020.06.004Get rights and content

Highlights

  • Cardiac CYLD is a mediator of pressure overload-induced cardiomyopathy.

  • Activation of cardiac autophagy is adaptive in pressure overloaded hearts.

  • ATG7 mainly promotes autophagic responses prior to the step of autolysosome efflux.

  • CYLD inhibits mTORC1 reactivation and autolysosome efflux in cardiomyocytes.

  • CYLD enables early autophagic adaptive responses to become detrimental to the heart.

Abstract

Deubiquitinating enzymes (DUBs) appear to be a new class of regulators of cardiac homeostasis and disease. However, DUB-mediated signaling in the heart is not well understood. Herein we report a novel mechanism by which cylindromatosis (CYLD), a DUB mediates cardiac pathological remodeling and dysfunction. Cardiomyocyte-restricted (CR) overexpression of CYLD (CR-CYLD) did not cause gross health issues and hardly affected cardiac function up to age of one year in both female and male mice at physiological conditions. However, CR-CYLD overexpression exacerbated pressure overload (PO)-induced cardiac dysfunction associated with suppressed cardiac hypertrophy and increased myocardial apoptosis in mice independent of the gender. At the molecular level, CR-CYLD overexpression enhanced PO-induced increases in poly-ubiquitinated proteins marked by lysine (K)48-linked ubiquitin chains and autophagic vacuoles containing undegraded contents while suppressing autophagic flux. Augmentation of cardiac autophagy via CR-ATG7 overexpression protected against PO-induced cardiac pathological remodeling and dysfunction in both female and male mice. Intriguingly, CR-CYLD overexpression switched the CR-ATG7 overexpression-dependent cardiac protection into myocardial damage and dysfunction associated with increased accumulation of autophagic vacuoles containing undegraded contents in the heart. Genetic manipulation of Cyld in combination with pharmacological modulation of autophagic functional status revealed that CYLD suppressed autolysosomal degradation and promoted cell death in cardiomyocytes. In addition, Cyld gene gain- and/or loss-of-function approaches in vitro and in vivo demonstrated that CYLD mediated cardiomyocyte death associated with impaired reactivation of mechanistic target of rapamycin complex 1 (mTORC1) and upregulated Ras genes from rat brain 7 (Rab7), two key components for autolysosomal degradation. These results demonstrate that CYLD serves as a novel mediator of cardiac pathological remodeling and dysfunction by suppressing autolysosome efflux in cardiomyocytes. Mechanistically, it is most likely that CYLD suppresses autolysosome efflux via impairing mTORC1 reactivation and interrupting Rab7 release from autolysosomes in cardiomyocytes.

Introduction

Since ubiquitin was discovered in the earlier 1970's, the ubiquitin proteasome system (UPS), which consists of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), proteasomes, and deubiquitinating enzymes (DUBs), has been implicated in virtually all aspects of cell biology [[1], [2], [3], [4], [5]]. Protein ubiquitination, a process in which ubiquitin (Ub) is covalently conjugated by its terminal glycine (G76) onto a lysine (K) residue of a substrate protein by the sequential action of an E1, E2 and E3, is a reversible posttranslational modification. A single Ub molecule may be attached, which is defined as monoubiquitination. Several lysine residues can be tagged with single Ub molecules, giving rise to multiple monoubiquitination, also referred to as multiubiquitination. Since Ub has seven lysine residues (K6, K11, K27, K29, K33, K48, K63) itself, Ub molecules can form different types of chains in an iterative process, known as polyubiquitination. In general, K48-linked poly-Ub chains represent a signal for proteasomal degradation of modified substrates, whereas mono-Ub and K63-linked poly-Ub chain modifications function as signaling devices for establishing protein-protein interactions and regulating cellular functions. The hydrolysis of ubiquitin linkage is conducted by DUBs. Eight E1s, a dozen different types of E2s, hundreds of E3s, and approximately 100 functional DUBs have been identified in humans.

During the past decades, an important role of Ub, E1s, several E2s and E3s, and proteasome in cardiac homeostasis and dysfunction has been well documented [6,7]. However, the myocardial function of DUBs is less well understood. Studies have demonstrated that A20 (also known as TNFAIP3) is a negative regulator of cardiac maladaptive remodeling and dysfunction via its ability to suppress the activation of nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs) and transforming growth factor beta (TGFβ) signaling [[8], [9], [10]]. Subsequent data have shown that cardiomyocyte-restricted (CR) overexpression of ubiquitin-specific protease (USP)15 results in age-dependent cardiac hypertrophy, indicating a mediator role of USP15 in cardiomyopathy [11]. Notably, our data showed that cylindromatosis (CYLD) [12] which has DUB activity highly specific for K63-linked Ub chains and is capable of suppressing NF-κB, MAPK and TGF-β signaling [13], is likely a crucial mediator of pressure overload (PO)-induced cardiomyopathy via a mechanism independent of the NF-κB pathway [14]. These findings underscore the functional specificity of individual DUBs in the heart, thereby supporting the notion that the association of DUBs with substrate adaptors, scaffolds and inhibitors may result in regulatory interactions that drive specificity [15,16]. Indeed, recent studies have revealed that USP4 and USP18 negatively regulate cardiac pathological remodeling [17,18] whereas USP14 may act as a mediator of cardiac pathological hypertrophy [19]. Nevertheless, the functional significance of individual DUBs in cardiac homeostasis and disease remains poorly understood.

Autophagy is an evolutionarily conserved catabolic process that targets cytoplasmic components such as organelles, protein aggregates or individual proteins to the lysosome for degradation. Autophagy in mammals has been classed into three types: (i) macroautophagy, (ii) microautophagy and (iii) chaperone-mediated autophagy (CMA). The macroautophagy (thereafter referred to as autophagy) is the best characterized. It is a very dynamic process that leads to formation of a double-membrane bound vesicle termed the autophagosome, which sequesters the targeted cargos, followed by autophagosome fusion with lysosomes to form the autolysosome, resulting in autolysosomal degradation (autolysosome efflux) [[20], [21], [22]]. The role of autophagy in the heart remains controversial. Depending the nature of stresses as well as the timing of assessments, activation of cardiac autophagy has been shown to be either adaptive or maladaptive [23]. In PO-hearts, recent studies have revealed that autophagy activation is most likely adaptive [20,23]. However, the definitive proof of autophagy-mediated cardiac protection in adult PO-hearts is still missing.

Recently, ubiquitination of substrates and protein components of autophagy machinery has emerged as a central regulatory mechanism of autophagy acting at various steps from autophagy induction to termination [24]. Therefore, it is not surprising that DUBs regulate autophagy [25]. Conceptually, it has been shown that otubain protease (OTUB)1, USP8, USP9X, USP10, USP13, USP19, USP20, and USP33 may act as positive regulators of autophagy, whereas A20, USP14, USP30, s-USP35, USP15, USP44, and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) probably serve as negative regulators of autophagy [25]. However, it is still not fully elucidated how the Ub code regulates autophagy in terms of altering protein signaling and targeting regulatory proteins for degradation. In addition, the crosstalk between E3s which add K63- or K48-linked poly-Ub chains to the components of autophagic machinery and DUBs that counteract their actions in the control of autophagy remains unclear. To date, the pathophysiological relevance of DUB-mediated regulation of autophagy in the heart has not yet been studied. For example, it is unclear whether the USP14- or USP15-mediated autophagy inhibition is linked to their pro-hypertrophic effects on the heart [11,18]. In addition, it remains unknown whether the A20-mediated autophagy inhibition documented in other pathways and processes such as self-directed immune responses [26] takes place in the heart as well.

In the present study, we demonstrated that CYLD suppresses autophagy at the stage of autolysosome efflux in cardiomyocytes, thereby exaggerating cardiac pathological remodeling and dysfunction in the setting of PO. These results uncovered a novel aspect of CYLD acting as an inhibitor of autophagy in the stressed hearts, thus providing the first evidence directly linking DUB-mediated autophagy regulation with cardiomyopathy.

Section snippets

Methods

Cardiomyocyte-restricted Cyld transgenic (CR-Cyld Tg) and CR-Atg7 × tTA Tg mice were generated as we previously reported [27,28]. Littermates of wild type (WT), CR-tTA Tg, CR-Atg7 Tg, CR-Cyld × tTA Tg, CR-Atg7 × tTA Tg, and CR-Cyld×Atg7 × tTA Tg mice were generated by crossbreeding between CR-Cyld Tg and CR-Atg7 × tTA Tg mice. Global Cyld knockout mice were generated as we previously reported [14]. Transverse aortic arch constriction (TAC) model, echocardiography, histopathology,

CR-CYLD overexpression exacerbates PO-induced cardiomyopathy

We have demonstrated that PO upregulates the expression of myocardial CYLD at both mRNA and protein levels and global KO of Cyld (CyldKO) protects against PO-induced cardiomyopathy, indicating a detrimental role of CYLD in the stressed hearts [14]. However, this notion remains to be verified by cardiac specific targeting of CYLD in vivo. Hence, we generated CR-Cyld Tg mice (Fig. S1A-E) and determined the impact of CR-CYLD overexpression on PO-induced cardiomyopathy in mice. At physiological

Discussion

In the present study, we demonstrate that CYLD is a mediator of PO-induced cardiomyopathy independent of gender differences. Mechanistically, CYLD suppresses autophagy by selectively interrupting autolysosome efflux in cardiomyocytes, thereby switching autophagic adaptation into autophagic damage in PO-hearts. At the molecular level, it is likely that CYLD inhibits mTORC1 reactivation and prevents Rab7 release from autolysosomes, both of which are required for autolysosome efflux in

Perspectives

Pressure overload initially enhances cardiac autophagy to protect against cardiac pathological remodeling and dysfunction. However, pressure overload over time increases the protein level of CYLD, leading to cardiac autophagy inhibition and contributing to cardiac maladaptive remodeling and dysfunction. At the molecular level, CYLD does not affect autophagosome biosynthesis and fusion with the lysosome but suppresses autolysosomal degradation after the autolysosome formation in cardiomyocytes.

Sources of funding

This research was supported by grants from the National Institute of Health (P01 AT003961, R01 HL131667) and American Diabetes Association (1–16-IBS-059), and a scholarship of China Scholarship Council (No. 201506220204).

Disclosures

None.

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