Attenuating PI3K/Akt- mTOR pathway reduces dihydrosphingosine 1 phosphate mediated collagen synthesis and hypertrophy in primary 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)
- et al.
Chapter 9 - drugs that inhibit signalling pathways for tumor cell growth and proliferation
Quantitative analysis of sphingoid base-1-phosphates as bisacetylated derivatives by liquid chromatography-tandem mass spectrometry
Anal. Biochem.
(2005)mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events
Cell
(2005)The Sphingosine-1-phosphate receptors S1P1, S1P2, and S1P3 function coordinately during embryonic angiogenesis*
J. Biol. Chem.
(2004)The Akt-SREBP nexus: cell signaling meets lipid metabolism
Trends Endocrinol. Metab.
(2010)- et al.
The inducible G protein-coupled receptor edg-1 signals via the G(i)/mitogen-activated protein kinase pathway
J. Biol. Chem.
(1996) Vertical inhibition of PI3K/Akt/mTOR signaling demonstrates in vitro and in vivo anti-fibrotic activity
J. Dermatol. Sci.
(2014)Exogenous Dihydrosphingosine 1 Phosphate Mediates Collagen Synthesis in Cardiac Fibroblasts Through JAK/STAT Signalling and Regulation of TIMP1
(2020)The akt-glycogen synthase kinase 3β pathway regulates transcription of atrial natriuretic factor induced by β-Adrenergic receptor stimulation in cardiac myocytes
J. Biol. Chem.
(2000)Glycogen synthase kinase 3β regulates GATA4 in cardiac myocytes
J. Biol. Chem.
(2001)
Revisiting the sphingolipid rheostat: evolving concepts in cancer therapy
Exp. Cell Res.
Pharmacological inhibition of PI3K reduces adiposity and metabolic syndrome in obese mice and Rhesus monkeys
Cell Metab.
Sphinganine-1-phosphate protects kidney and liver after hepatic ischemia and reperfusion in mice through S1P1 receptor activation
Lab. Invest.
EDG1 receptor stimulation leads to cardiac hypertrophy in rat neonatal myocytes
J. Mol. Cell. Cardiol.
Dihydroceramide desaturase 1, the gatekeeper of ceramide induced lipotoxicity
Biochimica et Biophysica Acta (BBA) – Mol. Cell Biol. Lipids
Sphingosine-1-phosphate: a Janus-faced mediator of fibrotic diseases
Biochim. Biophys. Acta
Collagen I and the fibroblast: high protein expression requires a new paradigm of post-transcriptional, feedback regulation
Biochem. Biophys. Rep.
Asiatic acid attenuates CCl4-induced liver fibrosis in rats by regulating the PI3K/AKT/mTOR and Bcl-2/Bax signaling pathways
Int. Immunopharmacol.
Targeting PI3K in cancer: mechanisms and advances in clinical trials
Mol. Cancer
Characterization and expression of sphingosine 1-Phosphate receptors in human and rat heart
Front. Pharmacol.
Disease model of GATA4 mutation reveals transcription factor cooperativity in human cardiogenesis
Cell
Phosphoinositide-3 kinase signaling in cardiac hypertrophy and heart failure
Curr. Pharm. Des.
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.
Sphingosine-1-Phosphate activates the AKT pathway to protect small intestines from radiation-induced endothelial apoptosis
Cancer Res.
Putting the brakes on cardiac hypertrophy
Hypertension
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
Sphingosine kinases and sphingosine 1-Phosphate receptors: signaling and actions in the cardiovascular system
Front. Pharmacol.
Sphingosine 1 phosphate receptor-1 (S1PR1) signaling protects cardiac function by inhibiting cardiomyocyte autophagy
J. Geriatr. Cardiol.
Novel insights into the role of HDL-Associated Sphingosine-1-Phosphate in cardiometabolic diseases
Int. J. Mol. Sci.
Serum sphingolipids level as a novel potential marker for early detection of human myocardial ischaemic injury
Front. Physiol.
Postinfarct left ventricular remodelling: a prevailing cause of heart failure
Cardiol. Res. Pract.
PI3K and calcium signaling in cardiovascular disease
Circ. Res.
Switching the sphingolipid rheostat in the treatment of diabetes and cancer comorbidity from a problem to an advantage
Biomed Res. Int.
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.
Glycogen synthase Kinase-3β is a negative regulator of cardiomyocyte hypertrophy
J. Cell Biol.
Cited by (22)
Lipid metabolism reprogramming in cardiac fibrosis
2024, Trends in Endocrinology and MetabolismHuman umbilical cord-derived stem cell sheets improve left ventricular function in rat models of ischemic heart failure
2022, European Journal of PharmacologyCitation Excerpt :Moreover, soluble endoglin inhibits TGF-β1-induced cardiac fibrosis and down-regulates the TGF-β1/Smads pathway to attenuate collagen production (Kapur et al., 2012). The PI3K/Akt/mTOR pathway is widely involved in the process of cardiac fibrosis and ventricular remodeling (Magaye et al., 2021; Yang et al., 2019). In this study, UC-MSC sheets inhibited the ratio of p-Akt/Akt and p-mTOR/mTOR, thus, showing that in the pathological changes of chronic ischemic HF, transplantation of UC MSCs inhibited the PI3K/Akt/mTOR pathway and improved cardiac function, suppressing ventricular remodeling.
Canagliflozin attenuates lipotoxicity in cardiomyocytes and protects diabetic mouse hearts by inhibiting the mTOR/HIF-1α pathway
2021, iScienceCitation Excerpt :In addition, although we had confirmed that mTOR could be a key target of CAN, other key factors in PI3K-AKT pathway also were predicated as the targets of CAN through molecular docking. Whether CAN also affects mTOR activity by PI3K-AKT pathway remains a matter further investigation because mTOR can be regulated by PI3K-AKT pathway (Magaye et al., 2021). Actually, CAN activated AKT in the previous study (Song et al., 2020).
Ovarian Tumor Domain-Containing 7B Attenuates Pathological Cardiac Hypertrophy by Inhibiting Ubiquitination and Degradation of Krüppel-Like Factor 4
2023, Journal of the American Heart AssociationThe role of sphingosine-1-phosphate in autophagy and related disorders
2023, Cell Death Discovery