Resident cardiac Sca-1-positive (+) stem cells may differentiate into cardiomyocytes to improve the function of damaged hearts. However, little is known about the inducers and molecular mechanisms underlying the myogenic conversion of Sca-1+ stem cells. Here we report that sphingosylphosphorylcholine (SPC), a naturally occurring bioactive lipid, induces the myogenic conversion of Sca-1+ stem cells, as evidenced by the increased expression of cardiac transcription factors (Nkx2.5 and GATA4), structural proteins (cardiac Troponin T), transcriptional enhancer (Mef2c) and GATA4 nucleus translocation. First, SPC activated JNK and STAT3, and the JNK inhibitor SP600125 or STAT3 inhibitor stattic impaired the SPC-induced expression of cardiac transcription factors and GATA4 nucleus translocation, which suggests that JNK and STAT3 participated in SPC-promoted cardiac differentiation. Moreover, STAT3 activation was inhibited by SP600125, whereas JNK was inhibited by β-cyclodextrin as a lipid raft breaker, which indicates a lipid raft/JNK/STAT3 pathway involved in SPC-induced myogenic transition. β-Catenin, degraded by activated GSK3β, was inhibited by SPC. Furthermore, GSK3β inhibitors weakened but the β-catenin inhibitor promoted SPC-induced differentiation. We found no crosstalk between the lipid raft/JNK/STAT3 and β-catenin pathway. Our study describes a lipid, SPC, as an endogenic inducer of myogenic conversion in Sca-1+ stem cells with low toxicity and high efficiency for uptake.

Cardiac stem cells (CSCs) have emerged as promising cell candidates to regenerate damaged hearts, because of the potential in differentiating to cardiomyocytes. However, the differentiation is difficult to trigger without inducers. Here we reported that β-cyclodextrin (β-CD) increased the expression of cardiac transcription factors (Nkx2.5 and GATA4), structural proteins (cardiac Troponin T, cTnt), transcriptional enhancer (Mef2c) and induced GATA4 nucleus translocation in adult resident CSCs, thus β-CD could be used to enhance myogenic transition. As the differentiation process was accompanied by autophagy, we constructed the Atg5 knockdown cell line by using the Atg5 siRNA lentivirus, and the myogenic conversion was blocked in Atg5 knockdown cells, which suggested that β-CD induces the cardiomyocytes transition of resident CSCs through autophagy. Furthermore, we found that JNK/STAT3 and GSK3β/β-catenin was the downstream pathways of β-CD-induced autophagy and differentiation using the inhibitors. Moreover, β-CD performed its functions through improving intracellular cholesterol levels and affecting cholesterol efflux. Collectively, our results reveal that β-CD as a novel tool to induce myogenic transition of CSCs, which could mobilize the resident CSCs or used together with CSCs to enhance the therapy effects of CSCs on damaged hearts. In addition, the clarified molecular mechanisms supported the new targets for inducing cardiomyocyte differentiation.