Figure 2.

The RRC is regulated by pyruvate dehydrogenase kinases. (a–c) Neonatal rat cardiac myocytes were incubated for 24 h in complete growth medium under normoxic (atmospheric O₂) (a) or hypoxic (<1% O₂) (b and c) conditions. After this period, RNA was either immediately extracted and subjected to qPCR for the indicated genes (b), or the medium was replaced with one that is glucose and fatty acid free, or one containing 17.5 mM glucose, 100 μM palmitate-BSA, or glucose+palmitate-BSA, as indicated, in normoxic conditions, for another 24 h (a and c). The results were averaged plotted as relative values to those from cells incubated in base medium in normoxic conditions adjusted to 1 (a and c) or control normoxia adjusted to 1 (b), n=3. Error bars represent standard error of the mean (S.E.M.), *P<0.05 versus control. (d) Neonatal cardiac myocyte was incubated for 24 h in complete growth medium and then infected with a control or adenoviruses (Ad) harboring PDK1, PDK4 or Hif-1α, for 24 h. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose plus 100 μM palmitate 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 control at the time point indicated; ^#P<0.05 max OCR for control versus max OCR of Pdk1 or Hif-1α treated. (e) Neonatal cardiac myocytes were cultured for 24 h in complete growth medium containing vehicle or 1 μM etomoxir, a Cpt1 inhibitor. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose plus 100 μM palmitate containing vehicle or 1 μM etomoxir, 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 control at the time point indicated; ^#P<0.05 max OCR for control versus max OCR for etomoxir treated, at the time point indicated (f and g). Neonatal rat cardiac myocytes were cultured in complete growth medium containing vehicle or 1 mM DCA and either remained in normoxic conditions (atmospheric O₂) (f) or were exposed to hypoxia (<1% O₂) (g), for 24 h. At the end of this period, the medium was changed to base medium containing 17.5 mM glucose or 17.5 mM glucose plus 100 μM palmitate-BSA containing vehicle or DCA, as indicated, for an additional 24 h. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose or 17.5 mM glucose plus 100 μM palmitate-BSA containing vehicle or 1 mM DCA, for 1 h. The mitochondrial stress test was then performed as described in Materials and Methods, n=4–6, each experiment was performed three times. 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 untreated, at the time point indicated