Figure 4.

The RRC in human iPSC-CM is regulated by DCA but not by AICAR. (a) Human iPSC-CM were cultured, fixed, permeabilized and incubated with the indicated primary antibodies. They were then incubated with fluorescently labeled phalliodin (upper panel) or secondary antibody (lower panel), mounted with DAPI and imaged by confocal microscopy. (b and c) Human iPSC-CM were cultured in complete growth medium containing vehicle, 1 mM DCA or 500 μM AICAR and either remained in normoxic conditions (atmospheric O₂) (b) or were exposed to hypoxia (<1% O₂) (c), for 24 h. At the end of this period, the medium was changed to base medium containing 17.5 mM glucose plus 100 μM palmitate-BSA containing vehicle, 1 mM DCA or 500 μM AICAR, as indicated, for an additional 24 h. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose plus 100 μM palmitate-BSA containing vehicle, 1 mM DCA or 500 μM AICAR, for 1 h. The mitochondrial stress test was then performed as described in Materials and Methods, n=4–6, each experiment was performed twice. Error bars represent S.E.M., *P<0.05 max OCR versus basal OCR for DCA-treated cells, at the time point indicated; ^#P<0.05 max OCR for DCA-treated versus max OCR for control, at the time point indicated