Here we used iPSCs to generate cardiomyocytes by reprogrammi

Here, we used iPSCs to generate cardiomyocytes by reprogramming skin fibroblasts from two patients carrying dissimilar LQTS mutations. Associated with LQT2, HERG-A422T produces trafficking-defective HERG K+ mpges-1 inhibitors (Kv 11.1) that decrease the rapidly activating delayed rectifier current (IKr), which is normally responsible for the bulk of ventricular repolarization. Conversely, NaV1.5-N406K produces a net gain of Na+ channel function, associated with LQT3. By simultaneously measuring membrane potential (Em) and intracellular free calcium ([Ca2+]i) transients, we compared the phenotypes of cardiomyocytes expressing these mutations. Our studies revealed similarly remodeled APs, with recurrent early afterdepolarizations (EADs) mirrored by comparable changes in [Ca2+]i transients. Such findings may point to a Ca2+-dependent, common arrhythmogenic mechanism, implying that a single therapeutic approach may be applicable to multiple forms of LQTS (Marbán, 2002; Roden and Viswanathan, 2005; Viskin, 1999).
Results
Discussion This investigation focused on identifying potential mechanisms underlying LQT arrhythmias as recapitulated in myocytes generated from patient-derived iPS cells. The results complement recent investigations showing that LQTS phenotypes can be produced in human stem cell-derived myocytes (Bellin et al., 2013; Davis et al., 2012; Itzhaki et al., 2011; Lahti et al., 2012; Terrenoire et al., 2013). Although arrhythmogenic triggers are recognized to be gene related, prolonged APs are the common denominator in LQTS diseases (Schwartz et al., 2001). Our results suggest that prolonged APs may promote arrhythmia, at least partially, through Ca2+-dependent mechanisms. Though similarities in profile between the AP and [Ca2+]i transient waveforms might not necessarily indicate a causal relationship, the results suggest that Em is influenced by three major Ca2+-dependent mechanisms in combination. First, blocking ICa strongly abbreviated the APs in LQT2 iPS-CM, even though AP prolongation had arisen from IKr deficiency. We attribute this to the normal presence of a sustained direct depolarization by ICa, and inward INCX during the triggered [Ca2+]i transient (Eisner et al., 1998; Linz and Meyer, 2000; Spencer and Sham, 2003). Second, stimulating SR Ca2+ release (with caffeine) during an ongoing AP prolonged its duration, with the resulting AP reflecting the [Ca2+]i waveform, and blocking SR Ca2+ reuptake (via SERCA) prolonged both APs and [Ca2+]i transients markedly. Thus, our findings support an established mechanism whereby a portion of released Ca2+ is extruded, generating inward INCX and thereby reactivating ICa and causing EADs (January et al., 1991; January and Riddle, 1989; Makielski and January, 1998). SR Ca2+ reuptake facilitates further cyclic Ca2+ releases during EADs, particularly when Em changes are relatively undamped by repolarizing currents—as in LQTS—(Figures S6E and S7). Third, in the presence of LiCl, cytosolic Ca2+ appeared to equilibrate between the SR and cellular buffers, made evident by the failure of [Ca2+]i to return to baseline despite membrane hyperpolarization possibly aided by limited Li+-Ca2+ exchange (Doering et al., 1998). When not blocked by LiCl, forward NCX would likely extrude this Ca2+ load as part of automaticity (Zahanich et al., 2011). Indeed, depleting SR Ca2+ by caffeine applications briefly reset spontaneous LQT2 [Ca2+]i transients to a shorter duration (Figure 4A). In both LQT2 and LQT3, the occasional prolongation of APs and Ca2+ transients out to tens of seconds also implies that mechanisms underlying the refractoriness and restitution of SR Ca2+ release are readily overridden in iPS-CM (Sobie et al., 2006). Taken together, our results show that in human LQTS myocytes (rather than drug-induced LQTS in animal models) Ca2+ handling is involved in prolonging the AP, regardless of the initiating mutation. Although we encourage additional studies examining a wider range of LQTS mutations, our results suggest that antiarrhythmic treatments for LQTS could target cellular Ca2+ cycling, with the benefit of being genotype independent.