Regulation of Stem Cell Differentiation Into Cardiomyocytes by Lysophosphatidic Acid

Abstract

The mechanisms that regulate the differentiation of stem cells (SCs) into cardiomyocytes are still unclear and the role of endogenous molecules on this process remains unexplored. One such molecule is the bioactive phospholipid lysophosphatidic acid (LPA) which accumulates in the myocardium following acute infarction and exerts multiple biological functions, including the regulation of cell growth and differentiation as well as cell survival (Tigyi et al., 2003; Sengupta, et al., 2004). Experiments were therefore carried out in this thesis to reveal whether LPA can induce the differentiation of stem cells into cardiomyocytes and to identify the signalling mechanisms that mediate this effect. All experiments were carried out in the mouse P19 carcinoma stem cell line. Treatments with LPA in the absence and presence of various pharmacological compounds were conducted in embryoid bodies (EBs) formed from the P19 cells in sterile Petri dishes over 4 days. The EBs were subsequently transferred into 6-well cell culture plates and cultured for specific time points. Lysates were generated and subjected to western blotting for expression of cardiac- specific myosin light chain -1v (MLC-1v). To look at the expression of LPA receptors (LPAR1-LPAR5) experiments were carried out by RT-PCR using specific primers for each LPA receptor and the role of the latter in mediated responses to LPA were examined in the presence of the LPAR 1/3 antagonist, Ki16425, or the LPAR 4 receptor blocker suramin. In addition, experiments were carried out investigating the role of Gαi and specific signalling pathways that may be involved in the differentiation of P19 cells. These were carried out using potent inhibitors/antagonists of Gi inhibitor (Pertussis toxin), PI3K inhibitor (LY294002), Akt inhibitor (Akt inhibitor XIII), PKC inhibitor (Bisindolylmaleimide I BIM-I), ROCK inhibitor (Y-27632), p38-MAPK inhibitor (SB203580) and ERK1/2 inhibitor (PD98059). Further experiments were carried out to establish whether the presence of LPA results in the phosphorylation of the targeted kinases. These studies were however limited to Akt, p38 MAPK and ERK1/2. Incubation of cells with LPA resulted in the differentiation of P19 cells into cardiomyocytes as reflected by the induction of MLC-1v. The latter increased significantly above basal in a time-dependent manner, reaching a maximum 10 days after plating EBs in 6-well plates. The induction of MLC-1v was more pronounced in cells incubated with 5 μM LPA at 6 days but showed little concentration differences at day 12. RT-PCR analysis confirmed the expression of LPA receptors 1 to 4 but not 5. Pre-incubating cells with suramin and Ki16425 concentration-dependently inhibited MLC-1v expression with 0.05 mg/ml and 10 M respectively, virtually abolishing the expression of MLC-1v. Additionally, inhibitors of LPAR1/3 and LPAR4 receptors and all the signalling inhibitors except SB203580 abolished the phosphorylation of ERK1/2. Similarly, p38 MAPK activation was completely abolished by LPAR1/3 and LPAR4 receptor antagonists, Interestingly, only LY294002 (5 M) and Y27632 (10 M) abolished the LPA induced activation of p38 MAPK while SB203580, BIM-I, Akt inhibitor XIII and PD95080 caused no significant changes to the phosphorylation of p38 MAPK. In conclusion, the studies carried out in this thesis have shown that LPA can induce P19 stem cells to differentiate into cardiomyocytes and they are linked to the well characterised LPA receptors (LPAR1/3 and 4). These receptors are coupled to downstream signalling pathways of which those involving the ROCK, PI3K, PKC and/or Akt may be critical, and may converge on ERK1/2. Inhibition of any of these pathways has the potential to suppress differentiation. In contrast, signalling leading to p38 activation may potentially suppress differentiation but this needs further clarification.