Genetic ablation and pharmacological inhibition of immunosubunit β5i attenuates cardiac remodeling in deoxycorticosterone-acetate (DOCA)-salt hypertensive mice

Highlights

• Ablation or inhibition of immunosubunit β5i attenuates DOCA-salt-induced adverse cardiac remodeling and function.

• Inhibition of β5i reduces PTEN degradation leading to inhibition of AKT/mTOR, TGF-β/Smad2/3, and NF-kB signaling.

• Blocking PTEN activity blunts the beneficial effect of β5i deletion on cardiac remodeling.

Abstract

Hypertensive cardiac remodeling is a major cause of heart failure. The immunoproteasome is an inducible form of the proteasome and its catalytic subunit β5i (also named LMP7) is involved in angiotensin II-induced atrial fibrillation; however, its role in deoxycorticosterone-acetate (DOCA)-salt-induced cardiac remodeling remains unclear. C57BL/6 J wild-type (WT) and β5i knockout (β5i KO) mice were subjected to uninephrectomy (sham) and DOCA-salt treatment for three weeks. Cardiac function, fibrosis, and inflammation were evaluated by echocardiography and histological analysis. Protein and gene expression levels were analyzed by quantitative real-time PCR and immunoblotting. Our results showed that after 21 days of DOCA-salt treatment, β5i expression and chymotrypsin-like activity were the most significantly increased factors in the heart compared with the sham control. Moreover, DOCA-salt-induced elevation of blood pressure, adverse cardiac function, chamber and myocyte hypertrophy, interstitial fibrosis, oxidative stress, and inflammation were markedly attenuated in β5i KO mice. These findings were verified in β5i inhibitor PR-957-treated mice. Moreover, blocking of PTEN (the gene of phosphate and tensin homolog deleted on chromosome ten) markedly attenuated the inhibitory effect of β5i knockout on DOCA-salt-induced cardiac remodeling. Mechanistically, DOCA-salt stress upregulated the expression of β5i, which promoted the degradation of PTEN and the activation of downstream signals (AKT/mTOR, TGF-β1/Smad2/3, NOX, and NF-κB), which ultimately led to cardiac hypertrophic remodeling. This study provides new evidence of the critical role of β5i in DOCA-salt-induced cardiac remodeling through the regulation of PTEN stability, and indicates that the inhibition of β5i may be a promising therapeutic target for the treatment of hypertensive heart diseases.

Introduction

Pathological cardiac remodeling is characterized by increased cardiomyocyte hypertrophy and death, myocardial fibrosis and contractile dysfunction of the heart, which lead to heart failure [1]. Various growth factors and mechanical stretch, including hypertension, angiotensin II (Ang II), and pressure or volume overload, can stimulate multiple signaling pathways that lead to hypertrophic remodeling [1]. Moreover chronic hypertension causes adverse cardiac function, hypertrophy and vascular remodeling. The mineralocorticoid aldosterone has been known to regulate sodium and water retention, and induce cardiac remodeling [2]. Deoxycorticosterone acetate (DOCA) is a synthetic mineralocorticoid derivative with a potent mineralocorticoid action. Chronic peripheral administration of DOCA combined with a high-sodium diet induces classic hypertension and cardiac remodeling with low-renin levels in different animals, thus it is regarded as an angiotensin-independent model [[3], [4], [5], [6]]. Moreover, DOCA-salt-treated animals display most of the changes of volume overload-induced hypertension in humans [3]. A growing body of data suggests that several signaling pathways contribute to DOCA-salt hypertension and cardiac remodeling, including PTEN/AKT/mTOR, SOCS3/JAK/STAT, TGF-β/Smad2/3, NADPH oxidase, and NF-κB [[4], [5], [6]]. However, the molecular mechanisms that regulate DOCA-salt induced cardiac remodeling have yet to be explored.

The proteasome, the main component of the ubiquitin-proteasome system (UPS), is an essential proteolytic complex for the degradation of ubiquitin-conjugated proteins [[7], [8], [9]]. The 26S proteasome contains two structures: the 20S core particle and the 19S regulatory particle. The core particle consists of two pairs of rings, and the proteolytic β subunits reside in the inner two rings. Among them, three standard catalytic subunits, including β1 (PMSB6), β2 (PMSB7), and β5 (PMSB5) are responsible for caspase-like, trypsin-like, and chymotrypsin-like activities, respectively [7]. When cells are stimulated by inflammatory stimuli such as IFN-γ, three of the standard catalytic subunits are replaced with three inducible subunits, including β1i (PMSB9 or LMP2), β2i (MECL-1, PMSB10, or LMP10), and β5i (PMSB8 or LMP7). This modified proteasome is called the immunoproteasome, which performs its proteolytic functions more efficiently than the standard proteasome [7]. Accumulating evidence indicates that the UPS plays a direct role in cardiac hypertrophy, apoptosis and sarcomere quality control, and is regulated by various hypertrophic stimuli such as Ang II, phenylephrine (PE), and pressure overload [6,10,11]. Our recent data revealed that the immunoproteasome catalytic subunit β2i is involved in the regulation of cardiac remodeling, atrial fibrillation (AF), and retinopathy in mice after DOCA-salt treatment or Ang II infusion [6,12,13]. More recently, we demonstrated a critical role of another immunosubunit (β5i) in the development of Ang II-induced AF and abdominal aortic aneurysm (AAA) [14,15]. However, the functional role of β5i in the development of DOCA-salt-induced cardiac remodeling remains unclear.

In this study, our results indicated for the first time that DOCA-salt treatment significantly increases the expression of β5i and chymotrypsin-like activity in the heart. The ablation and inhibition of β5i markedly reduced DOCA-salt-induced hypertension, cardiac remodeling, and dysfunction. Moreover, blocking PTEN activity with a specific inhibitor (VO-Ohpic) markedly abolished the inhibitory effect of β5i deletion on cardiac remodeling in mice. Thus, our results demonstrated that the inhibition of β5i prevents DOCA-salt-induced cardiac remodeling by blocking PTEN degradation and the activation of downstream mediators.