We set out to develop a way to instruct the

We set out to develop a way to instruct the ESCs to differentiate into a cardiac pacemaker subtype with a factor relevant to embryonic pacemaker development. Native cardiac pacemaker mass calculator are anatomically confined in the sinoatrial node (SAN), a diminutive structure comprising just a few thousand genuine pacemaker cells (Bleeker et al., 1980). During embryonic development, cardiac pacemaker cells originate from a subset of progenitors distinct from the first (marked by Nkx2.5) and second (marked by Isl1) heart fields not only in their genetic makeup (Christoffels et al., 2010; Wiese et al., 2009), but also in their anatomic location (Bressan et al., 2013). However, an area of Hcn4-positive primordial SAN is reported to express Isl1 (Mommersteeg et al., 2007), suggesting that second heart field progenitors may also contribute to the developing SAN. We have recently demonstrated that postnatal re-expression of an embryonic transcription factor, Tbx18, converts ventricular cardiomyocytes to pacemaker cells, recapitulating morphological as well as electrophysiological hallmarks of genuine SAN pacemaker cells (Kapoor et al., 2013). Elsewhere, transgenic overexpression of Tbx3 has been shown to elicit ectopic rhythm in mouse atrial myocardium (Bakker et al., 2012). Noting the powerful capacity of embryonic transcription factors in determining the fate of cardiac cell subtype, we hypothesized that overexpression of a SAN-specific transcription factor may steer ESC differentiation toward pacemaker cell subtype. Here, we report that heterologous expression of SHOX2 during early stages of mouse ESC (mESC) differentiation strongly favors a SAN-specific gene program, leading to enhanced pacemaker cell specification. The differentiated cells exhibit greater automaticity in vitro and perform biological pacemaker function when injected into the rat heart in vivo.
Results
Discussion Shox2 is indispensable for proper formation and development of the SAN. Shox2 null/null mouse embryos exhibit severe hypoplasia of the SAN accompanied by an aberrant expression of chamber cardiomyocyte-specific markers in the SAN (Blaschke et al., 2007; Espinoza-Lewis et al., 2009). Mouse Shox2 is first detected at ED8.5 in the posterior region of the developing heart tube, and its expression terminates by ED 13.5, being restricted to the cardiac conduction system (Blaschke et al., 2007; Espinoza-Lewis et al., 2009). Genetic ablation of Shox2 results in slowed contraction rate in mESC-derived EBs (Hashem et al., 2013). Here, we demonstrate that transient and heterologous expression of SHOX2 greatly increases the percentage of spontaneously beating EBs, the number of beating foci in each EB, and the EB contraction rate compared with control (Figures 4A–4D). The enhanced automaticity correlates directly with more HCN4+ cells present in SHOX2-EBs compared with control (Figure 3A). Direct injection of SHOX2-EBs into the rat left ventricular apex created ectopic automaticity indicative of induced biological pacing at a rate faster than the junctional escape rhythm observed in hearts injected with GFP-EBs (Figure 4E). The enhanced automaticity is accompanied by increased expression of automaticity-promoting genes and gene products such as HCN4, NCX1, and CX45 (Figure 3). As rationale for the timing of SHOX2 overexpression, it may have been more logical to base it when Shox2-positive progenitors exist. In this regard, Shox2 expression is detected as early as day 6 of differentiation (Hashem et al., 2013) in an mESC-derived EB system. This largely coincides with the time points of exogenous SHOX2 overexpression (D3, D6, D7) in our study. It is notable that the beating rates of the GFP-EBs rise and then recede between D6+9 and D6+11 (Figure 4C). The majority of embryonic cardiac myocytes have the tendency to beat spontaneously but become more quiescent as they mature electrophysiologically (Pelleg et al., 1980). Likewise, the rapid decrease in the beating rates of GFP-EBs may be due to changes in their electrophysiological components, similar to the swift increase in INa density in differentiating, mESC-derived EBs (Maltsev et al., 1994). In contrast, the spontaneous beating rates remain high in SHOX2-EBs (Figure 4C), further supporting the notion that the superior automaticity of SHOX2-EBs is due to enhanced differentiation to pacemaker cells rather than to nonspecific deterioration of chamber cardiomyocytes.