MicroRNA-365 accelerates cardiac hypertrophy by inhibiting autophagy via the modulation of Skp2 expression

https://doi.org/10.1016/j.bbrc.2017.01.108Get rights and content

Highlights

  • Enhanced expression of miR-365 in hypertrophic cardiomyopathy.

  • Reduction of LC3-II, LC3-II/I ratio and beclin-1 expression in hypertrophic cardiomyopathy.

  • MiR-365 promotes cardiac hypertrophy and inhibits autophagy.

  • MiR-365 inhibits the expression of Skp2 in hypertrophied cardiomyocytes.

  • Skp2 restrains cardiac hypertrophy by promoting autophagy.

Abstract

Evidence is emerging of a tight link between cardiomyocyte autophagy and cardiac hypertrophy (CH). Sustained exposure to stress leads CH to progress to heart failure. Several miRNAs have been described in heart failure, and miRNA-based therapeutic approaches are being pursued. Although microRNA-365 (miR-365) has been testified as a positive modulator of CH, the specific mechanism remains unclear. In the present study, we observed that miR-365 expression was up-regulated in hypertrophic cardiomyocytes both in vivo and in vitro, and was accompanied by dysregulation of autophagy. We found that miR-365 negatively modulates autophagy in hypertrophic cardiomyocytes by targeting Skp2. Overexpression of Skp2 promoted autophagy and rescued CH induced by Ang-II; conversely, Skp2 knockdown further inhibited autophagy and CH. Furthermore, we found that the activation of mammalian target of rapamycin (mTOR) signaling was regulated by Skp2 following Ang-II treatment, as indicated by the up-regulation of p-S6K and p-4EBP1 levels. The inactivation of mTOR by rapamycin completely abolished the Ang-II-induced inhibition of autophagy. In conclusion, our study provides substantial evidence that miR-365 and its target gene Skp2 play a functional role in CH and suggests the development of novel therapeutic options based on miR-365 and Skp2.

Introduction

Pathological cardiac hypertrophy (CH) is a primary risk factor for almost all forms of heart failure and is characterized by enlargement of cardiomyocytes, enhanced protein synthesis, up-regulation of fetal genes and disruption of sarcomeric structure [1]. During the compensatory stage of CH, the increase in cardiomyocyte size and mass is thought to be involved in biomechanical, physiological, structural and metabolic changes that take place in order to maintain heart function. However, under prolonged chronic stress or disease will result in irreversible ventricular dilation, a decline in contractile function, cardiac de-compensation and eventually progression to heart failure [2]. Further investigation of the mechanisms underlying pathological CH would promote the development of novel therapeutic strategies for the treatment of heart failure.

There is increasing evidence that miRNAs play an essential role in the pathogenesis of various cardiac diseases, including cardiac concentric hypertrophy, viral myocarditis, ischemia-reperfusion injury and heart failure [3], [4]. Previous studies suggested that the process of pathological CH is closely associated with the dysregulation of many miRNAs both in vitro and in vivo [3], [5]. MiR-365 has been reported to be involved in the myocardial regenerative process in neonatal mouse heart and neointimal formation in atherosclerosis patients [6], [7]. Furthermore, overexpression of miR-365 alone is able to increase the size of cardiomyocytes [8]. However, the specific underlying mechanisms of miR-365 in the progression of CH have not been well characterized.

Autophagy, as a highly conserved catabolic process, is necessary to balance cellular energy metabolism via degradation, the recycling of redundant cytoplasmic proteins and quality control of intracellular organelles [9]. Autophagy plays essential and complicated roles in the progression of cardiac remolding and heart failure under various pathological stimuli. On the one hand, autophagy can act as a protective process that promotes myocardial cell survival through degradation and recycling of proteins and damaged organelles; on the other hand, autophagy may serve as a maladaptive response that triggers cell death due to the excessive degradation of organelles under stress stimulation [10]. The abrogation of continuous constitutive autophagy owing to an Atg5 deficiency results in spontaneous CH and disorders in cardiac structure and function [11]. Proper autophagy was involved in functional homeostasis of the cardiovascular system and was affected by several miRNAs; recently, autophagy has been identified to be regulated by miR-221/miR-222, which inhibit the progression of CH and subsequent heart failure [5], [12]. To date, whether miR-365 modulates CH progression by regulating autophagy in cardiomyocytes remains unknown.

In this study, we aimed to investigate the specific molecular role of miR-365 in autophagy and progression of CH. We found that the cardiac-specific up-regulation of miR-365 is involved in the progression of CH both in vivo and in vitro. Owing to the inhibition of autophagy induced by miR-365, the volume of myocardial cells increased. We also found that miR-365 modulates autophagy in CH by decreasing Skp2 and hyper-activating mTORC1 signaling under the induction of CH.

Section snippets

Animals

Male adult C57BL6 mice (SPF, 10 weeks old) were purchased from Vitalriver Laboratory Animal Company (Beijing, China). All experimental procedures were conducted in line with the principles approved by the institutional Animal Care and Use Committee. A mouse model of CH was induced via transverse aortic constriction (TAC) as previously described [13]. Mice were deeply anesthetized using ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed in a supine position. Then, a 5 mm section of the

Enhanced expression of miR-365 in hypertrophic cardiomyopathy in vivo

We first detected the expression of miR-365 in a mouse model of CH. The results showed that the induction of hypertrophic cardiomyopathy led to an apparent up-regulation of hypertrophic responses, as determined by increased heart weight/body weight ratio (Fig. 1A), and the hypertrophic markers atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) (Fig. 1B and C) (P < 0.05). More importantly, the expression of miR-365 was remarkably up-regulated in the heart of mice subjected to

Discussion

Abnormal expression of miRNAs in response to various biomechanical and pathophysiological stimuli plays an essential role in detrimental CH and subsequent heart failure, which is a leading clinical cause of high morbidity and mortality [2], [19]. Herein, we found that overexpression of miR-365 accelerates CH, as evidenced by the remarkable up-regulation of miR-365 in both hypertrophic mouse heart and cultured cardiomyocytes subjected to Ang-II treatment, accompanied by enhanced expression of

Conflict of interest statement

We would like to submit the enclosed manuscript entitled “MicroRNA-365 accelerates cardiac hypertrophy by inhibiting autophagy via the modulation of Skp2 expression”, which we wish to be considered for publication in your honored journal. No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published

References (28)

  • H.L. Liu et al.

    Identification of the microRNA expression profile in the regenerative neonatal mouse heart by deep sequencing

    Cell Biochem. Biophys.

    (2014)
  • M.H. Kim et al.

    MicroRNA-365 inhibits the proliferation of vascular smooth muscle cells by targeting cyclin D1

    J. Cell Biochem.

    (2014)
  • Z. Li et al.

    Functions of autophagy in pathological cardiac hypertrophy

    Int. J. Biol. Sci.

    (2015)
  • Y. Matsui et al.

    Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy

    Circ. Res.

    (2007)
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