Attenuating PI3K/Akt- mTOR pathway reduces dihydrosphingosine 1 phosphate mediated collagen synthesis and hypertrophy in primary cardiac cells

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Abstract

Cardiac fibrosis and myocyte hypertrophy play contributory roles in the progression of diseases such as heart Failure (HF) through what is collectively termed cardiac remodelling. The phosphoinositide 3- kinase (PI3K), protein kinase B (Akt) and mammalian target for rapamycin (mTOR) signalling pathway (PI3K/Akt- mTOR) is an important pathway in protein synthesis, cell growth, cell proliferation, and lipid metabolism. The sphingolipid, dihydrosphingosine 1 phosphate (dhS1P) has been shown to bind to high density lipids in plasma. Unlike its analog, spingosine 1 phosphate (S1P), the role of dhS1P in cardiac fibrosis is still being deciphered. This study was conducted to investigate the effect of dhS1P on PI3K/Akt signalling in primary cardiac fibroblasts and myocytes. Our findings demonstrate that inhibiting PI3K reduced collagen synthesis in neonatal cardiac fibroblasts (NCFs), and hypertrophy in neonatal cardiac myocytes (NCMs) induced by dhS1P, in vitro. Reduced activation of the PI3K/Akt- mTOR signalling pathway led to impaired translation of fibrotic proteins such as collagen 1 (Coll1) and transforming growth factor β (TGFβ) and inhibited the transcription and translation of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). PI3K inhibition also affected the gene expression of S1P receptors and enzymes such as the dihydroceramide delta 4 desaturase (DEGS1) and sphingosine kinase 1 (SK1) in the de novo sphingolipid pathway. While in myocytes, PI3K inhibition reduced myocyte hypertrophy induced by dhS1P by reducing skeletal muscle α- actin (αSKA) mRNA expression, and protein translation due to increased glycogen synthase kinase 3β (GSK3β) mRNA expression. Our findings show a relationship between the PI3K/Akt- mTOR signalling cascade and exogenous dhS1P induced collagen synthesis and myocyte hypertrophy in primary neonatal cardiac cells.

Introduction

Remodelling in the injured myocardium is characterised by increased activation of hypertrophic and fibrotic factors, and has detrimental effects in progressive heart failure (HF) (Galli and Lombardi, 2016). Years of research have linked many peptide hormones, growth factors, cytokines, lipid mediators, and their downstream effectors to the process of cardiac remodelling (Schirone et al., 2017; Nabben et al., 2018). Research by our group and others have implicated sphingolipids, S1P and its analog dhS1P, in cardiac fibrosis and hypertrophy (Magaye et al., 2020; Schwalm et al., 2013; Robert et al., 2001; Ohkura et al., 2017). These lipids are ubiquitously present in biological systems and function as lipid mediators. In circulation they are bound to HDL and albumin (Mishima et al., 2018; Diarte-Añazco et al., 2019). In human platelet depleted plasma, their concentration ranges from 0.1 to 0.6 μM, with dhS1P at the lower end (Berdyshev et al., 2005). Their levels are altered in plasma, erythrocytes and platelets of patients with cardiomyopathies (Knapp et al., 2012a; Jadczyk et al., 2018; Egom et al., 2013). They interact with the sphingosine 1 phosphate receptors (S1PRs) on cell membranes, which are G protein coupled receptors (Robert et al., 2001; Means and Brown, 2009), leading to activation of downstream signalling pathways such as the phosphatidylinositol 3- kinase/ Protein Kinase B (PI3K/ Akt) pathway.

Lipid Kinases such as the PI3Ks are increasingly being targeted for cancer and immune disorder therapies (Yang et al., 2019). PI3Ks are composed of three different classes of enzymes involved in cellular function. Class 1 PI3K activates Akt, which serves as the main downstream effector of PI3K. Among other substrates, Akt then phosphorylates mTOR, which acts on other proteins such as RPS6 to increase ribosomal RNA, nuclear protein synthesis, growth regulatory gene product, and cyclin D (Avendaño and Menéndez, 2008). Together with the other broad range of cascading signals, activation of the PI3K/Akt pathway contributes to tissue and organ fibrosis, cell proliferation and survival (Wei et al., 2018; Hsu et al., 2017; Liu and Zhuang, 2016; Liang et al., 2014; Ghigo et al., 2017). PI3K/ Akt also plays an important role in cellular lipid metabolism (Ortega-Molina et al., 2015; Krycer et al., 2010).

Although S1P is known to activate the PI3K/Akt pathway in other cell types (Liu and Tie, 2019; Bonnaud et al., 2010; Wang et al., 2018a), the effect of S1P and its analogue, dhS1P, on the PI3K/ Akt pathway in primary cardiac myocytes and fibroblast is less understood. Considering the plethora of molecular and cellular events that contribute to collagen synthesis in myocyte hypertrophy in pathological events such as cardiac remodelling, here we investigate the effects of PI3K/ Akt- mTOR signalling pathway activation by dhS1P and S1P in isolated primary neonatal rat cardiac fibroblasts and myocytes.

Section snippets

Methods

Primary NCMs and NCFs were extracted from 1 to 2 days old Sprague-Dawley rat pups using enzymatic collagenase digestion routinely used in our laboratory for in vitro assays (Lekawanvijit et al., 2010). Pups were purchased from the Monash Animal Research Platform (Clayton, Vic), and complied with the guidance from the National Health and Medical Research Council of Australia. The animals used for this study was approved by the Alfred Medical Research and Education Precinct Animal Ethics

Inhibition of PI3K led to reduction of dhS1P & S1P induced collagen synthesis

One of the hallmarks of fibrosis in tissues including the heart is exacerbated collagen synthesis by cells such as fibroblasts. Our previous studies have shown that dhS1P and S1P at doses of 3 μM and 1 μM induced collagen synthesis in primary NCFs (Magaye et al., 2020). Here we investigated whether this increased collagen synthesis could be reduced by inhibiting the PI3K/Akt pathway, using a potent PI3K inhibitor (W- wortmannin). Our results in Fig. 1 show that wortmannin was able to inhibit

Discussion

In this study we investigated the role of the PI3K/Akt- mTOR pathway in terms of collagen synthesis and myocyte hypertrophy mediated by the sphingolipids, dhS1P and S1P, in primary neonatal rat cardiac fibroblasts and myocytes. S1P has been shown to be cardioprotective by a number of researchers in cases of ischemia reperfusion injury and S1P signalling through S1PR1 in cardiac myocytes has also been shown to reduce myocyte hypertrophy (Chen et al., 2018; Cannavo et al., 2013). However, our

Conclusion

The PI3K/Akt- mTOR signalling pathway plays a complex role in dhS1P and S1P induced cardiac fibrosis and myocyte hypertrophy. In terms of collagen synthesis in cardiac fibroblasts, these findings indicate that inhibition of PI3K/Akt – mTOR signalling cascade in NCFs treated with dhS1P has anti- proliferative and anti- fibrotic effects by reducing translation of growth factors and increased degradation of ECM. Although, our findings are preliminary, they show links between the PI3K/ Akt- mTOR

Funding

This research was supported by National Health and Medical Research Council of Australia (Program Grants 1092642 and Project Grant 1087355.

RM and FS were supported by the Monash University International Post Graduate Research Scholarships and the Monash Graduate Scholarships.

CRediT authorship contribution statement

Ruth R. Magaye: Data curation, Formal analysis, Methodology, Writing - original draft, Writing - review & editing. Feby Savira: Data curation, Formal analysis, Writing - original draft. Yue Hua: Data curation, Formal analysis. Xin Xiong: Data curation, Formal analysis, Writing - review & editing. Li Huang: Data curation, Formal analysis. Christopher Reid: Writing - review & editing. Bernard L. Flynn: Writing - review & editing. David Kaye: Writing - review & editing. Danny Liew: Writing -

Declaration of competing interest

The authors declare no conflict of interest.

References (70)

  • J. Newton

    Revisiting the sphingolipid rheostat: evolving concepts in cancer therapy

    Exp. Cell Res.

    (2015)
  • A. Ortega-Molina

    Pharmacological inhibition of PI3K reduces adiposity and metabolic syndrome in obese mice and Rhesus monkeys

    Cell Metab.

    (2015)
  • S.W. Park

    Sphinganine-1-phosphate protects kidney and liver after hepatic ischemia and reperfusion in mice through S1P1 receptor activation

    Lab. Invest.

    (2010)
  • P. Robert

    EDG1 receptor stimulation leads to cardiac hypertrophy in rat neonatal myocytes

    J. Mol. Cell. Cardiol.

    (2001)
  • S. Rodriguez-Cuenca et al.

    Dihydroceramide desaturase 1, the gatekeeper of ceramide induced lipotoxicity

    Biochimica et Biophysica Acta (BBA) – Mol. Cell Biol. Lipids

    (2015)
  • S. Schwalm et al.

    Sphingosine-1-phosphate: a Janus-faced mediator of fibrotic diseases

    Biochim. Biophys. Acta

    (2013)
  • R.I. Schwarz

    Collagen I and the fibroblast: high protein expression requires a new paradigm of post-transcriptional, feedback regulation

    Biochem. Biophys. Rep.

    (2015)
  • L. Wei

    Asiatic acid attenuates CCl4-induced liver fibrosis in rats by regulating the PI3K/AKT/mTOR and Bcl-2/Bax signaling pathways

    Int. Immunopharmacol.

    (2018)
  • J. Yang

    Targeting PI3K in cancer: mechanisms and advances in clinical trials

    Mol. Cancer

    (2019)
  • N. Ahmed

    Characterization and expression of sphingosine 1-Phosphate receptors in human and rat heart

    Front. Pharmacol.

    (2017)
  • Y.-S. Ang

    Disease model of GATA4 mutation reveals transcription factor cooperativity in human cardiogenesis

    Cell

    (2016)
  • T. Aoyagi et al.

    Phosphoinositide-3 kinase signaling in cardiac hypertrophy and heart failure

    Curr. Pharm. Des.

    (2011)
  • K.M. Argraves

    S1P, dihydro-S1P and C24:1-ceramide levels in the HDL-containing fraction of serum inversely correlate with occurrence of ischemic heart disease

    Lipids Health Dis.

    (2011)
  • S. Bonnaud

    Sphingosine-1-Phosphate activates the AKT pathway to protect small intestines from radiation-induced endothelial apoptosis

    Cancer Res.

    (2010)
  • W. Booz George

    Putting the brakes on cardiac hypertrophy

    Hypertension

    (2005)
  • A. Cannavo

    beta1-adrenergic receptor and sphingosine-1-phosphate receptor 1 (S1PR1) reciprocal downregulation influences cardiac hypertrophic response and progression to heart failure: protective role of S1PR1 cardiac gene therapy

    Circulation

    (2013)
  • A. Cannavo

    Sphingosine kinases and sphingosine 1-Phosphate receptors: signaling and actions in the cardiovascular system

    Front. Pharmacol.

    (2017)
  • Y.-Z. Chen

    Sphingosine 1 phosphate receptor-1 (S1PR1) signaling protects cardiac function by inhibiting cardiomyocyte autophagy

    J. Geriatr. Cardiol.

    (2018)
  • E.M.G. Diarte-Añazco

    Novel insights into the role of HDL-Associated Sphingosine-1-Phosphate in cardiometabolic diseases

    Int. J. Mol. Sci.

    (2019)
  • E.E. Egom

    Serum sphingolipids level as a novel potential marker for early detection of human myocardial ischaemic injury

    Front. Physiol.

    (2013)
  • A. Galli et al.

    Postinfarct left ventricular remodelling: a prevailing cause of heart failure

    Cardiol. Res. Pract.

    (2016)
  • A. Ghigo

    PI3K and calcium signaling in cardiovascular disease

    Circ. Res.

    (2017)
  • N.K. Haass et al.

    Switching the sphingolipid rheostat in the treatment of diabetes and cancer comorbidity from a problem to an advantage

    Biomed Res. Int.

    (2015)
  • K.M. Hannan

    mTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF†

    Mol. Cell. Biol.

    (2003)
  • S. Haq

    Glycogen synthase Kinase-3β is a negative regulator of cardiomyocyte hypertrophy

    J. Cell Biol.

    (2000)
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