Enrichment of cardiac differentiation of mouse pluripotent stem cells by beta 3 adrenoceptor stimulation

Background: Identification of signaling mechanisms able to enrich cardiac differentiation of stem cells is important to envisage cardiac stem cell therapy. It is previously shown that β-adrenoceptors (β-Rs) stimulation through β1 and β2-Rs enhances cardiomyogenesis of mouse embryonic stem (mES) cells and survival of cardiac progenitor cells (CPCs). In heart failure (HF) it is established that β1 and β2-Rs have different effects on cardiomyocytes compared to β3-R. A recent clinical trial showed that β3-Rs stimulation by mirabegron increased left ventricular ejection fraction (LVEF) in patients with severe chronic HF. Purpose: Despite β3-R role in adult heart is extensively studied, no data exist on β3-Rs during cardiac maturation. Therefore, we investigated the role of β3-Rs during cardiac maturation analysing structural, molecular and functional phenotypes of Embryoid Bodies (mEBs) differentiated from mESC and the effect of β3-Rs signaling modulation. Methods: mES were differentiated into the cardiogenic lineage in control conditions (Ctr) and with BRL37344 (7µM) or SR59230A (10µM), selective β3-Rs activator or inhibitor, respectively. Results: WB analysis showed that throughout cardiac differentiation β1-R was highly expressed in the early phase and significantly declining thereafter, while β3 have a rather stable expression during the process. q-PCR revealed that spontaneous contractile activity of mEBs was modulated by β3-R: BRL37344 treatment significantly increases expression level of cardiogenic genes, including mesodermal (d5: brachyury ≈+760%), precardiac (d5: Wnt3A ≈+25000%, Wnt11 ≈+20000% and β-catenin ≈+725%), first heart field (d7: Mef2C ≈+1360%, Hand1 ≈+1000%) and late cardiac markers (d7: Gata4 ≈+60%, Nkx2.5 ≈+62%, Tbx5 ≈+44%, Tbx20 ≈+150%). Oppositely, SR59230A reducing significantly the expression of mesodermal (d5: brachyury ≈-50%, Mesp1 ≈-96%), precardiac (d5: Wnt3A ≈-99%, Wnt11 ≈-88%), first (d7: Mef2C ≈-90%, Hand1 ≈-95%), second heart field (d7:hand2 ≈-99%, Isl1 ≈-84%, Tbx1 ≈-71%, Fgf10 ≈-90%) and late cardiac markers (d7: Gata4 ≈-90%, Nkx2.5 ≈-95%, Tbx5 ≈-95%, Tbx20 ≈-95%). Moreover, frequency of spontaneous contraction was significantly enhanced by β3-R stimulation (d12: from 27±0.6 to 37±1.4bpm), suggesting that electrogenic mechanisms driving mES spontaneous beating may be regulated by β3-R signaling. We further explored this point by analysis of genes involved in sinoatrial node (SAN) formation (d7: Tbx3≈ 1100%, Tbx18≈+7000% and Shox2≈+4200%), development (d7: cTnI≈+5700%, Cav1.3≈+5700%) and function (d8: Hcn1≈+7000%, Hcn4≈+1300%) and observed that β3-R stimulation causes a robust up-regulation. Finally, atrial and ventricular myosin light chains genes expression (d7: mlc2a≈+3100% and mlc2v≈+280%) confirmed a shifted toward atrial phenotype. Conclusion. β3-Rs are functionally expressed in mES undergoing cardiogenic differentiation and mediate an important signal to enable cardiac specification of mES.