Inositol-1,4,5-trisphosphate-mediated spontaneous activity in mouse embryonic stem cell-derived cardiomyocytes.

In early embryonic heart as well as in the adult sinoatrial (SA) node, spontaneous pacemaker activity strongly depends on the presence of Ca2+ signalling mechanisms (Sakai et al. 1983; Komuro et al. 1985; Bogdanov et al. 2001; Lipsius et al. 2001). In the early embryonic heart, spontaneous activity was shown to be initiated by Ca2+ influx, whereas in the rabbit or cat SA node, the rhythmic increase in spontaneous ryanodine receptor (RyR)-mediated Ca2+ release events and subsequent activation of the Na+/Ca2+ exchanger (NCX) seem to be the driving force for the diastolic depolarization (Huser et al. 2000; Bogdanov et al. 2001). One reason for the differences in the pacemaker mechanism could be the developmental changes in the Ca2+ handling mechanisms. While voltage-dependent L-type Ca2+ channels are expressed from day 9.5 days post coitum (dpc) (Takemura et al. 2005), T-type channels have been observed around embryonic day 14 (Cribbs et al. 2001). Influx through L-type channels is thought to be the major source of Ca2+ for excitation–contraction coupling (ECC) in early embryonic cardiomyocytes, whereas the main source of Ca2+ in adult cells during ECC is RyR-mediated Ca2+-induced Ca2+ release (CICR) (Escobar et al. 2004; Perez et al. 2005). The cause for this seems to be the rudimentary development of the sarcoplasmic reticulum (SR) and the low expression of RyRs (Gorza et al. 1997; Rosemblit et al. 1999; Escobar et al. 2004). The role of inositol-1,4,5-trisphosphate (IP3) receptor (IP3R)-mediated Ca2+ release in cardiac muscle is still debated. Recent studies have suggested a role in the generation of arrhythmic activity in atrial and ventricular myocytes or in the defined activation of cardiac transcription factors (Zima & Blatter, 2004; Proven et al. 2006; Wu et al. 2006). However, it seems that IP3-mediated Ca2+ release through IP3R channels may play a more prominent role in embryonic cardiomyocytes. IP3 production has been linked to a specific perinuclear Ca2+ release and shown to regulate mitochondrial Ca2+ uptake in neonatal rat cardiomyocytes (Jaconi et al. 2000). Embryonic stem cells differentiate in vitro into spontaneously active multicellular cardiomyocyte preparations while recapitulating developmental stages of the early embryonic cardiomyogenesis (Boheler et al. 2002; Hescheler et al. 2002; Banach et al. 2003). They express cardiac-specific marker proteins (e.g. αMHC and MLC-2v) and transcription factors (e.g. Nkx-2.5, MEF2C and GATA-4) (Narita et al. 1997; Boheler et al. 2002; Wobus & Boheler, 2005). Shortly after the onset of spontaneous synchronized beating, the proliferation of the embryonic stem cell-derived cardiomyocytes (ESdCs) ceases and the multicellular aggregates expand by cell growth (Meyer et al. 2000; Banach et al. 2003; He et al. 2003; Puceat et al. 2003). Intracellular recordings from individual ESdCs dissociated from aggregates of beating cells show that different cellular phenotypes such as sinus node-like, atrial-like and ventricular-like cells develop (Maltsev et al. 1999; Abi-Gerges et al. 2000; Fijnvandraat et al. 2003; He et al. 2003). With the in vitro differentiation of ESdCs, expression of the Ca2+-handling proteins such as RyR (cardiac type-2 isoform), NCX1, sarcoplasmic reticulum Ca2+ ATPase (SERCA)2 and phospholamban has been described (Boheler et al. 2002; Fijnvandraat et al. 2003). Furthermore, the following voltage-dependent currents have been shown in ESdCs: If (Gryshchenko et al. 1999; Abi-Gerges et al. 2000), Ito and Isus (Gryshchenko et al. 2000), and INa, ICa,T and IL,Ca (Abi-Gerges et al. 2000; Zhang et al. 2003). Pacemaker activity in ESdC aggregates, connexin expression and conduction velocity also follow closely the development of the embryonic heart (Banach et al. 2003) and pacemaker frequency is responsive to the neurohumoral stimuli isoprenaline and acetylcholine (Ji et al. 1999; Abi-Gerges et al. 2000; Banach et al. 2003). The spontaneous activity in early embryonic cardiomyocytes is thought to be significantly regulated by If (Satoh & Sperelakis, 1993; Robinson et al. 1997); however, it also depends critically on the presence of Ca2+ in the extracellular solution (Fujii et al. 1981a,Fujii 1981b). It has been proposed that intracellular Ca2+ oscillations in ESdCs are independent of voltage-activated Ca2+ influx (Viatchenko-Karpinski et al. 1999; Mery et al. 2005). RyR-mediated Ca2+ release has been demonstrated by the presence of Ca2+ sparks and caffeine transients in ESdCs (Sauer et al. 2001) and RyR-mediated Ca2+ release was shown to contribute to their pacemaker activity (Fu et al. 2006). However, spontaneous whole-cell Ca2+ transients continue in the presence of caffeine and in ESdCs deficient for Ry R2 expression (Sauer et al. 2001; Yang et al. 2002). By contrast, specific blocking or down-regulation of IP3R type 1 suppresses Ca2+ signalling and automaticity of ESdCs and embryonic heart tubes (Mery et al. 2005). Until now, neither the basis for IP3R activation nor the mechanism by which IP3R-mediated Ca2+ release results in the spontaneous whole-cell Ca2+ transients of the ESdCs has been determined. In the present study, we tested the hypothesis that the developmental changes in the Ca2+ handling mechanisms of ESdCs modulate the frequency of their spontaneous activity, and that IP3-mediated Ca2+ release needs to be translated into a depolarization of the membrane potential (Vm) and subsequent activation of voltage-dependent Ca2+ channels to generate a whole-cell Ca2+ transient.