AMP-activated protein kinase α1-sensitive activation of AP-1 in cardiomyocytes

https://doi.org/10.1016/j.yjmcc.2016.04.009Get rights and content

Highlights

  • Cardiac Ampkα1 is upregulated by pressure overload and angiotensin-II infusion.

  • Ampkα1 activates the transcription factor AP-1 via Pkcζ in cardiomyocytes.

  • AP-1 activation by angiotensin-II is blunted by silencing of Ampkα1 but not Ampkα2.

  • Cardiac Ampkα isoform shift may contribute to the signaling of cardiac remodeling.

Abstract

AMP-activated protein kinase (Ampk) regulates myocardial energy metabolism and plays a crucial role in the response to cell stress. In the failing heart, an isoform shift of the predominant Ampkα2 to the Ampkα1 was observed. The present study explored possible isoform specific effects of Ampkα1 in cardiomyocytes. To this end, experiments were performed in HL-1 cardiomyocytes, as well as in Ampkα1-deficient and corresponding wild-type mice and mice following AAV9-mediated cardiac overexpression of constitutively active Ampkα1. As a result, in HL-1 cardiomyocytes, overexpression of constitutively active Ampkα1 increased the phosphorylation of Pkcζ. Constitutively active Ampkα1 further increased AP-1-dependent transcriptional activity and mRNA expression of the AP-1 target genes c-Fos, Il6 and Ncx1, effects blunted by Pkcζ silencing. In HL-1 cardiomyocytes, angiotensin-II activated AP-1, an effect blunted by silencing of Ampkα1 and Pkcζ, but not of Ampkα2. In wild-type mice, angiotensin-II infusion increased cardiac Ampkα1 and cardiac Pkcζ protein levels, as well as c-Fos, Il6 and Ncx1 mRNA expression, effects blunted in Ampkα1-deficient mice. Pressure overload by transverse aortic constriction (TAC) similarly increased cardiac Ampkα1 and Pkcζ abundance as well as c-Fos, Il6 and Ncx1 mRNA expression, effects again blunted in Ampkα1-deficient mice. AAV9-mediated cardiac overexpression of constitutively active Ampkα1 increased Pkcζ protein abundance and the mRNA expression of c-Fos, Il6 and Ncx1 in cardiac tissue. In conclusion, Ampkα1 promotes myocardial AP-1 activation in a Pkcζ-dependent manner and thus contributes to cardiac stress signaling.

Introduction

The Adenosine 5′-monophosphate (AMP)-activated protein kinase (Ampk) is activated by an increase of the cytosolic AMP/ATP concentration ratio and of the cytosolic Ca2 + concentration [1]. The heterotrimeric structure of Ampk consists of a catalytic alpha (α) subunit and regulatory beta (β) and gamma (γ) subunits [2]. The two isoforms of the catalytic α subunit show a different cellular localization: Ampkα1 is predominantly found in the non-nuclear fraction and Ampkα2 is found in both the nuclear and the non-nuclear fraction [3]. The Ampkα1 isoform is ubiquitously expressed, whereas the Ampkα2 isoform is mainly expressed in skeletal muscle and heart [2].

The two catalytic Ampkα isoforms may differ in activating mechanisms, targets and effects [4], [5], [6]. In contrast to Ampkα2, Ampkα1 is implied as an important mediator of inflammatory signals [7]. In skeletal muscle, a shift towards an increased Ampkα1 activity is observed following iron deficiency [8]. Myogenesis is promoted by Ampkα1, but not by Ampkα2 [9]. Ampkα1 is required for macrophage skewing to an M2 phenotype during muscle regeneration [10]. Ampkα1 further stimulates Na+/K+ ATPase endocytosis and alveolar epithelial dysfunction [11].

Ampk is considered to play an important role in cardiac ischemia, hypertrophy and failure [2]. Ampk is activated in cardiac tissue under physiological and pathological stress [2]. Cardiomyocytes predominately express the Ampkα2 isoform [2]. In the failing human heart, an isoform shift from the Ampkα2 to the Ampkα1 subunit is observed [12], [13]. In rat cardiac hypertrophy induced by pressure overload, an increased Ampk activity is paralleled by reduced Ampkα2 but increased Ampkα1 expression [14]. Also obstructive nephropathy induces an isoform shift from Ampkα2 towards Ampkα1, which participates in renal stress signaling [15].

Loss of Ampkα2 results in exacerbation of pressure overload-induced cardiac dysfunction in mice [16]. Ampkα1, on the other hand, is required for proliferation of cardiac fibroblasts induced by angiotensin-II [17]. Furthermore, Ampkα1 promotes scar formation and myofibroblast activity after chronic cardiac ischemia [18]. Ampkα1 further affects gap junction remodeling following pressure overload, an effect apparently also involved in cardiac ischemia [19], [20]. Ampkγ2 subunit mutations are associated with cardiac hypertrophy, electrophysiological abnormalities and glycogen storage disease [21].

In lymphocytes, Ampkα1 activates the transcription factor activator protein-1 (AP-1) [22], [23], an important regulator of cardiomyocyte function involved in cardiac remodeling [24], [25], [26]. In failing hearts, AP-1 activity is increased [27] and results in up-regulation of c-Fos, interleukin-6 (Il6) and Na+/Ca2 + exchanger (Ncx1) [28], [29], [30]. Signaling regulating AP-1 activity includes protein kinase C zeta (Pkcζ) [31]. Stimulators of AP-1 transcriptional activity include angiotensin-II [32].

The present study therefore addressed possible direct effects of Ampkα1 on myocardial stress signaling. The effects of Ampkα1 on AP-1 activation as downstream effector were investigated in HL-1 cardiomyocytes with and without angiotensin-II treatment. To investigate the possible effects of the cardiac Ampkα isoform shift during cardiac remodeling in vivo, Ampkα1-deficient mice (Ampkα1−/−) and corresponding wild-type mice (Ampkα1+/+) were treated with angiotensin-II to mimic neurohumoral activation and pressure overload by transverse aortic constriction (TAC) to induce mechanical stress. Effects of Ampkα1 in cardiac tissue were further investigated in mice following AAV9-mediated cardiac overexpression of constitutively active Ampkα1.

Section snippets

Materials and methods

A detailed description of the methods can be found as online supplemental information.

Results

A first series of experiments explored whether the Ampkα1 isoform is involved in the regulation of cardiac Pkcζ. To this end, HL-1 cardiomyocytes were transfected with a construct encoding constitutively active Prkaa1T183D (CAα1) or with the empty vector as control. Pkcζ phosphorylation at Thr410 was measured in Pkcζ-immunoprecipitated samples. In view of the low abundance of Pkcζ, immunoprecipitation was employed to avoid isoform cross-reaction of the phospho-Pkcζ/λ (Thr410/403) antibody. No

Discussion

In accordance with previous observations, the strained heart shows an isoform shift with increasing expression of the Ampkα1 isoform [13], [19], [33]. Increased Ampk activity has been described during pressure overload [13], [14]. Both, neurohumoral activation by angiotensin-II and mechanical stress by pressure overload increased Ampkα1 abundance. AAV9-mediated cardiac overexpression of constitutively active Ampkα1 for 4 weeks did not lead to hypertrophy and did not appreciably modify cardiac

Disclosures

The authors declare that they have no conflicts of interest with the contents of this article.

Acknowledgements

The authors gratefully acknowledge Dr. W.C. Claycomb for providing the HL-1 cardiomyocyte cell line and Dr. B. Viollet for providing the Ampkα1−/− mouse. The authors are grateful for the outstanding technical assistance of E. Faber and K. Streil and the meticulous preparation of the manuscript by T. Loch and A. Soleimanpour.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (La315/4-5 and SFB-Transregio 19), the German Cardiac Society (DGK Stipendium 2012) and the

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