Mechanical signals play a crucial regulatory role in cardiac

Mechanical signals play a crucial regulatory role in cardiac growth, development, and maintenance (Happe and Engler, 2016). Here, we developed a bioreactor system that exposes ESC-derived tizanidine hcl to defined pulsatile flow and cyclic strain, thereby mimicking in vivo physical signals that are important for normal cardiac development (Andrés-Delgado and Mercader, 2016). Compared with other recent bioreactor-related studies, which focused on either using cyclic strain or cyclic strain and electrical stimulation (Torsoni et al., 2005; Gwak et al., 2008; Tulloch et al., 2011; Huang et al., 2012; Nunes et al., 2013; Mihic et al., 2014), we highlight the importance of combining pulsatile flow and cyclic stain to drive ESC-CM maturation in vitro. The applied shear stresses induced by pulsatile flow in this study (∼10−2 to 10−3 dyn/cm2) differ from physiological blood luminal flow (∼10–20 dyn/cm2) (Butcher and Nerem, 2007; Chiu and Chien, 2011), but instead correspond to the in vivo environment, where CMs are exposed to an extremely low transmural flow rather than direct shear stress (Andrés-Delgado and Mercader, 2016). Of note, cardiac wall strains display significant temporal and regional variations (−7.9% ± 3.8% to +11.3% ± 6.4% in vivo [Tsamis et al., 2011]). To mimic cardiac strain, previous studies applied cyclic strain between 2.5% and 12% to PSC-CMs, and demonstrated that cyclic strain conditioning can increase the expression of cardiac-associated markers and improve organization of sarcomere proteins (Shyu et al., 2010; Huang et al., 2012; Mihic et al., 2014). In accordance with these studies, culturing mESC-derived cells for 12 days in the presence of cyclic strains alone led to a significant upregulation of cardiac-associated genes and an increased number of MF20+ cells when compared with static controls. We further observed a synergistic effect of the combination of 1.48 mL/min pulsatile flow with 5% cyclic strain, which led to a significant increase of cardiac-associated gene expression and an improved alignment of sarcomeric fibers. When then extending the culture time, markers associated with CM maturation were further increased in both murine and human ESC-CMs. In detail, d18 dyn mESC-CMs and d20 dyn hESC-CMs displayed well-organized sarcomeric proteins, a higher gap junction protein expression, and an increased cardiac ion channel gene expression. d20 dyn hESC-CMs exhibited an average sarcomere length of 1.97 ± 0.25 μm, which is higher than the average sarcomere length of hESC-CMs reported in other studies and similar to the sarcomere length of relaxed adult CMs (Borg et al., 2000; Feinberg et al., 2013; Nunes et al., 2013). Maturation of ESC-CMs is often accompanied by increased ion channel expression. Our data showed an increase in relevant ion channel genes in d18 dyn mESC-CMs and d20 dyn hESC-CMs when compared with d18 stat mESC-CMs or d20 stat hESC-CMs (normalized to either Gapdh [mouse]/GAPDH [human], or Rplp0 [mouse]/RPLP0 [human]). Raman microspectroscopy was employed in a previous study for the marker-free characterization of different CM phenotypes (Brauchle et al., 2016). Here, we compared Raman spectra and identified that structural protein- and lipid-related peaks were more prominent in d18 dyn mESC-CMs when compared with d18 stat mESC-CMs. The stronger protein-related peaks in d18 dyn mESC-CMs had been previously identified as glycogen (Pascut et al., 2011, 2013). A higher presence of glycogen in ESC-CMs was previously attributed to an increased glycolytic metabolism in CMs, which is required to produce myofibril contractions (Pascut et al., 2011, 2013). Lipids have been described to play an important role in energy storage and homeostasis in cardiac muscle (Chung et al., 2007). The increase in lipid-related bands in d18 dyn mESC-CMs might be attributed to a metabolic shift toward β-oxidation of fatty acids (Chung et al., 2007; Brauchle et al., 2016). The comparison of mESC-CMs and primary isolated fCMs and aCMs revealed that d18 dyn mESC-CMs exhibited a phenotype closer to murine aCMs. Similar to murine aCMs, human aCMs exhibit a lower nuclear and higher mitochondrial density as well as a metabolic shift (Pohjoismaki et al., 2013). In accordance, d20 dyn hESC-CMs and human fCMs showed stronger structural protein- and lipid-related peak intensities, but decreased signals of nucleotide bands when compared with hESC-CMs cultured for only 10 days or under static conditions. The observed differences in protein-assigned bands are possibly due to the sarcomeric organization and increasing myofibril densities in the d20 dyn hESC-CMs, as was confirmed by IF staining in this study and as hypothesized previously (Brauchle et al., 2016). These findings highlight the importance of combined pulsatile flow and cyclic strain in the maturation process of PSC-CMs.