Since mitochondria are a principal source of ROS production

Since mitochondria are a principal source of ROS production, we also investigated whether PGC-1α-dependent mitochondrial changes result in altered ROS levels in cardiomyocytes. Although PGC-1α can induce the expression of antioxidant enzymes (St-Pierre et al., 2006), in this context the impact of having a greater number of mitochondrial ROS-producing sites must outweigh the induction of detoxification pathways, because ROS levels were increased. Remarkably, upon PGC-1α knockdown, the amplitude and duration of the AP and the maximum amplitude of the calcium transient were increased to values closer to those found in adult cardiomyocytes, despite a marked energetic defect. Energetic compromise in cardiomyocytes typically shortens the AP as a result of increased ATP-regulated potassium current, and decreases the calcium transient amplitude (Baartscheer et al., 2011; Koumi et al., 1997; Nichols et al., 1991). Yet, counter to this, there is evidence for an inhibitory effect of oxidative stress on the calcium current and AP duration (Goldhaber et al., 1989; Guerra et al., 1996). Given our evidence for the oligomycin insensitivity (at least upon short-term exposure) of the AP in these early cardiomyocytes, the benefit of lowered oxidative stress may be dominant in determining the overall outcome. Remarkably, repression of the mitochondrial biogenesis program in these Birinapant resulted in an improvement in the calcium transient. Fortunately, however, the ROS levels could be lowered directly in control cardiomyocytes with a combination of culture at physiological oxygen tension and antioxidant supplementation, resulting in an improvement in the calcium transient to the same degree as PGC-1α knockdown. Based on these data, it may be worthwhile to make this a standard aspect of PSC-derived cardiomyocyte culture, at least at later stages of differentiation. Although an increase in mitochondrial mass is a feature of maturity, if the ROS produced by the mitochondria are not controlled, the overall function and contractility of the cardiomyocytes may be restricted. Further work will be required to determine precisely how ROS should be manipulated temporally to control the balance between maturation-related mitochondrial biogenesis and wider cell function. Increasing the energetic demands of the cardiomyocytes through stimulation with FCS, and exerting physiologically relevant chronotropic stimulation through beta-adrenergic receptor activation both exposed shortcomings in PGC-1α knockdown cardiomyocytes. FCS induced a consistent hypertrophic growth, and in cells that had repressed PGC-1α or were uncoupled by DNP, this resulted in sarcomeric disorganization, presumably as a multifactorial response to inadequate energy supply. The mechanisms underlying the PGC-1α-dependent loss of automaticity upon isoproterenol stimulation, as well as the lower AP frequency under basal conditions, are unknown. They may relate to the longer AP we observed in these cells, the chronic energy disturbance, or an unknown connection between PGC-1α and automaticity. In line with this, both PGC-1α and PGC-1β knockout mouse hearts show blunted responses to beta-adrenergic stimulation with dobutamine, and it has been suggested they may be required for maximal automaticity of pacemaker cells, although a mechanism is lacking (Arany et al., 2005; Lelliott et al., 2006; Leone et al., 2005). We set out to develop a human model of acquired heart disease by diminishing mitochondrial function. The rationale for this stemmed from the large body of literature reporting downregulation of mitochondrial pathways during heart failure, but with little explanation for how and why this occurs, or what consequences it has for individual heart cells (Ventura-Clapier et al., 2011). We confirmed that hESC-derived cardiomyocytes are a valuable tool for exploring the functional relationships between mitochondria and heart disease, revealing perhaps unexpected outcomes. Such studies go hand in hand with the goal of producing more adult-like cells for these and other applications. To maximize the potential function of these cells, it is important to control ROS, since the mitochondrial biogenesis program is activated in PSC-derived cardiomyocytes.