Cardiac phosphorylation of protein kinase B/Akt and downstream proteins after bupivacaine toxicity. (A) Cardiac output as measured by rate pressure product (RPP = mean arterial pressure × heart rate) in response to bupivacaine toxicity. (B) Theoretical cardiac bupivacaine (solid line) concentration with 95% CI (dotted lines) based on radiolabel studies (see Materials and Methods: Bupivacaine Concentration Fit Curves and Grouping). (C) Relative RPP difference between groups above ionotropic cation channel (e.g., sodium and calcium) IC₅₀ (greater than 100 μM bupivacaine) and below cation channel IC₅₀ (less than 100 μM bupivacaine), *P < 0.05. (D) Western blots of cardiac lysates at different time points during recovery for phosphoproteins in the insulinergic pathway including protein kinase B (Akt) phosphorylated at S473; Akt phosphorylated at T308; total Akt, glycogen synthase kinase-3α (GSK-3α) and -3β (GSK-3β) phosphorylated at S21 and S9, respectively; p70 s6 kinase (p70s6k) phosphorylated at T421; ribosomal protein s6 (s6) phosphorylated at S235, insulin receptor substrate-1 (IRS1) phosphorylated at S612; and total glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. (E) Densitometry of cardiac lysates (from “D”) comparing control phosphorylation (n = 4) to unrecovered animals with greater than 100 μM of cardiac bupivacaine (n = 4); *P < 0.05, **P < 0.01, ***P < 0.001, Sidak post hoc test.