Mitochondrial ROS generation following acetylcholine-induced EGF receptor transactivation requires metalloproteinase cleavage of proHB-EGF

Acetylcholine (ACh) mimics ischemic preconditioning by a mechanism dependent on phosphatidylinositol 3-kinase (PI3-kinase) and reactive oxygen species (ROS). In other tissues muscarinic receptors activate a metalloproteinase, which liberates surface-associated heparin-binding epidermal growth factor (HB-EGF) and causes transactivation of epidermal growth factor receptors (EGFRs) with activation of PI3-kinase. We tested whether this pathway is operative in myocardium. Adult rabbit cardiomyocytes were incubated in reduced MitoTracker Red, which fluoresces after ROS exposure. ACh caused a 36 ± 6% increase in fluorescence (P < 0.001) and metalloproteinase inhibitor III (MPI) abolished this increase. Both exogenous EGF as well as HB-EGF caused similar increases in the ROS signal (41 ± 12%, P = 0.005 and 40 ± 7%, P < 0.001, respectively). The ROS burst from HB-EGF was unaffected by MPI (37 ± 6%, P = 0.002), confirming that inhibition of metalloproteinase activity blocked ACh’s effect at a site upstream of EGFR. CRM-197, which inhibits HB-EGF activity, also blocked ACh-induced ROS generation, again implicating release of HB-EGF as a necessary step for ROS generation. An HB-EGF-neutralizing antibody also prevented ACh-induced increase in ROS. In isolated, perfused rabbit hearts ACh increased phosphorylation of EGFR by 127.4 ± 43.7%, and this increase was abolished by MPI. Finally, ACh decreased infarct size from 30.1 ± 2.9% of the risk zone in control hearts to 13.7 ± 3.0% (P = 0.002), and this protection could be abolished by co-treatment with MPI (28.7 ± 2.6%, P = n.s. vs. control). Stimulation of a second Gi-protein-coupled receptor by the δ-opioid agonist [D-Ala2, D-Leu5]-enkephalin acetate (DADLE) also protected the heart (9.1 ± 2.0% infarction, P < 0.005 vs. control), and this protection was similarly blocked by MPI (28.9 ± 2.3% infarction). We conclude that ACh-induced ROS generation in myocytes is mediated via transactivation of EGFR through metalloproteinase-dependent release of HB-EGF, and that this pathway is also operative in the intact heart and is required for ACh’s cardioprotection.