Neuregulin/erbB signalling is cardioprotective as seen in models of cardiotoxicity associated with the use of chemotherapeutics. Our studies reveal a function of Bcl-2 to protect cardiomyocytes concomitant with GGF2-induced increases in levels of phospho-BAD (pBAD) along with activation of PI3 kinase (PI3K) and MAP kinase (MAPK). We show that phosphorylation of BAD is dependent upon GGF2 mediated activation of PI3K. In addition, examination of mitochondrial gene expression shows several members of the Bcl-2 family are down-regulated by doxorubicin, while GGF2 is able to reverse this effect. While many in vitro model systems of doxorubicin cardiotoxicity have observed PI3K-dependent cytoprotective effects of neuregulin's, the present results demonstrate that GGF2 mediates cytoprotective actions in HL-1 cardiomyocytes through regulation of mitochondrial mediated apoptosis via the PI3K/Bcl-2 pathway.

Glial growth factor (GGF2) is a Neuregulin-1 (NRG-1), which binds ErbB receptors and activates downstream cell signaling processes. NRG-1 ligands and their receptors are required for cardiac development and adult cardiac function. GGF2 has been shown to improve left ventricular function in several models of heart failure though the mechanisms by which GGF2 is efficacious are poorly understood. GGF2 is currently in clinical trials for treatment of congestive heart failure. Many studies have focused on GGF2 regulation of PI3 kinase (PI3K) as measured by formation of pAKT in vivo and in numerous cell lines, including HL-1 cells, neonatal rat ventricular myocytes (NRVMs) and human iPSC-derived cardiomyocytes. NRG-1s also activate the MAP kinase (MAPK) pathway as measured by pERK1/2 formation. To better understand GGF2 action in the stressed heart, we have developed in vitro systems to study GGF2 protection of cardiomyocytes from doxorubicin induced toxicity. After serum starvation, cells were pre-incubated with GGF2 (0.15 pM to 9.5 nM) for 1 hr followed by exposure to 1 μM doxorubicin for 18 hrs. After doxorubicin treatment MTT labeling index of cultures was used as an endpoint measurement of cell metabolic status. Doxorubicin decreases MTT labeling index by 90% while GGF2 prevented this toxicity by almost 50% compared to untreated cells, with an EC 50 of 83.3±19.4 pM . This effect appears to be dependent upon AKT signaling and not MAPK as assessed using inhibitors of both pathways. Inhibition of MAPK increases pAKT formation without changing extent of GGF2 protection. Blocking ErbB4 receptor, highly expressed in cardiomyocytes, causes a decrease in pAKT formation, suggesting that this receptor is important for mediating GGF2 action in these cells. Our results demonstrate that, in HL-1 cells, GGF2 mediates cytoprotective responses which require PI3K activation. Similar studies are being pursued in doxorubicin-treated NRVMs and human iPS derived cardiomyocytes. Initial results indicate GGF2 also mediates cytoprotective effects in these cell types. Future efforts will be aimed at elucidating potential roles for PI3K-dependent GGF2 regulation of mitochondrial function, Ca 2+ signaling or REDOX regulation in the observed cytoprotective actions.