Cytoskeletal and rhythmic contraction and pulsatile laminar shear stress are

Cytoskeletal and rhythmic contraction, and pulsatile laminar shear stress, are mechanical forces that have been suggested to play a crucial role in heart development and growth (Taber, 2001; Zhu et al., 2014; Andrés-Delgado and Mercader, 2016). Cytoskeletal contraction is involved in several morphogenetic processes in the embryo (Taber, 2001). Rhythmic contraction is the exposure of CMs to regular cyclic stretch (Zhu et al., 2014). The heart is exposed to blood flow during most heart developmental stages and throughout adult life (Andrés-Delgado and Mercader, 2016). Pulsatile laminar shear stress generated by blood flow in healthy hearts influences heart chamber formation and maturation, trabeculation, CM proliferation, and valvulogenesis (Andrés-Delgado and Mercader, 2016). Employing physical signals to mimic cardiogenesis in vitro is a potential strategy to achieve PSC-CM maturation. Several groups have developed methods to apply uniaxial stress to PSCs (Tulloch et al., 2011; Wan et al., 2011; Mihic et al., 2014). Although uniaxial stress can promote PSC-CM maturation, using stretch alone does not mimic in vivo physical signals that act on CMs within the heart (Guan et al., 2011). Carrier et al. (2002) demonstrated that perfusion flow leads to a continuous medium change and therefore increases the spatial uniformity of CMs by improving the control of oxygen, pH, nutrients, and metabolites in the cellular microenvironment; however, the impact of pulsatile flow on PSC-CM maturation, or a possible synergistic effect of the combination of pulsatile flow and cyclic strain has not been investigated.
In the present study, we designed and evaluated a bioreactor system to expose mouse embryonic stem cell (mESC)- and human ESC (hESCs)-derived filipin to defined mechanical stimuli. We investigated the impact of pulsatile flow-induced shear stress and physiological stretch on murine and human ESC-CM maturation in vitro by extensively analyzing cardiac protein and gene expression patterns. We analyzed the calcium-handling properties and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity in dynamically-cultured CMs. In addition, we characterized the phenotype of ESC-CMs employing filipin marker-free Raman microspectroscopy. Elucidating the effect of defined mechanical forces on the maturation of ESC-CMs is an essential step toward developing fully matured and functional cardiovascular tissues in vitro that can be used to study cardiovascular diseases and investigate potential drug candidates.

Results

Discussion
In our study, we revealed that d18 dyn mESC-CMs and d20 dyn hESC-CMs showed a functional improvement as the typical systolic calcium transient parameters, the rate of constant of Ca2+ decay (tau), slope, and amplitude improved with extended culture times, indicating rapid electromechanical coupling. This change could be the result of structural changes in the contractile apparatus, the better orientation of the myocyte network associated with enhanced expression of gap junctions, or alterations in the balance of cardiac ion channels (Zhu et al., 2014). In previous studies, most of the ESC-CMs did not respond to caffeine as they relied on calcium influx from sarcolemma instead of the SR as seen in adult CMs (Khan et al., 2013; Li et al., 2013). In a few studies, hESC-CMs cultured for more than 27 days showed a response to caffeine (Nunes et al., 2013; Kosmidis et al., 2015). Here, ESC-CMs responded to caffeine after 12 (mESC-CMs) or 10 days (hESC-CMs), as shown by an increased Ca2+ handling consistent with a functional SR. Furthermore, when compared with d12 dyn mESC-CMs and d10 hESC-CMs, d18 dyn mESC-CMs and d20 dyn hESC-CMs exhibited a higher store of Ca2+ and an improved SERCA function. We further performed concentration-response studies employing nifedipine and dofetilide. Although hESC-CMs exhibited a dose-dependent response to nifedipine and dofetilide, no significant differences were seen between stat and dyn hESC-CMs. The electrophysiology was stable throughout the recordings. This indicates that the calcium and hERG channels in all hESC-CMs were fully functional without any limitation.