Sex differences in the mechanism of Met5-enkephalin-induced cardioprotection: role of PI3K/Akt

Abstract

Met(5)-enkephalin (ME)-induced cardioprotection occurs via epidermal growth factor receptor (EGFR) transactivation with the subsequent activation of phosphatidylinositol 3-kinase (PI3K). In the present study, we investigated whether there is a sex difference in ME-elicited PI3K signaling. Neonatal murine cardiomyocytes were isolated by collagenase digestion and subjected to 90 min hypoxia and 180 min reoxygenation at 37 degrees C (n = 5 to 7 replicates). PI3K/Akt signaling was interrogated using pharmacological inhibitors and small interfering RNA (siRNA). Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. The data are presented as means +/- SE. There was not a sex difference in the basal content of total Akt. ME (100 microM) elicited comparable protection in both sexes. Wortmannin and the nonselective Akt inhibitor IV completely abolished ME-induced protection in male cardiomyocytes but only attenuated protection in female cardiomyocytes. Isoform-selective knockdown of Akt in males with siRNAs against Akt1/2 completely abolished ME-induced cardioprotection, whereas the siRNAs against Akt3 only attenuated protection of approximately 40%. In contrast, in females the siRNAs against Akt1/2 attenuated and against Akt3 eliminated ME-induced cardioprotection. There is not a sex difference in the degree of ME-induced protection, and there is a sex difference in the cardioprotective signaling pathways after the administration of ME; ME-induced cardioprotection in males primarily utilizes a PI3K/Akt1/2 pathway and in females primarily utilizes a PI3K/Akt3 pathway. The incomplete loss of protection in females following the blockade of PI3K suggests that additional factors may facilitate the maintenance or function of activated Akt.

Fig. 1

Competitive inhibition of phosphatidylinositol 3-kinase (PI3K) with LY-294002 (LY) abolishes Met⁵-enkephalin (ME)-induced cardioprotection in males and attenuates protection in females. AUC, area under the curve; NS, not significant. Data are expressed as means ± SE.

Fig. 2

Irreversible inhibition of PI3K with wortmannin (Wort) also abolishes ME-induced cardioprotection in males and attenuates protection in females. Data are expressed as means ± SE.

Fig. 3

Pharmacological inhibition of pan-Akt eliminates ME-induced protection in males and attenuates cardioprotection in females. Data are expressed as means ± SE.

Fig. 4

Basal and stimulated Akt content. There was no difference in basal (unstimulated) content of total Akt between males and females. However, administration of ME resulted in a maximal ∼20% increase in phosphorylated to total Akt in male cardiomyocytes at 15 min, whereas ME elicited ∼200% increase in phosphorylation of Akt in female cardiomyocytes at 15 min. Data are expressed as means ± SE. p/t, Phosphorylated/total; au, arbitrary units.

Fig. 5

Akt knockdown by small interfering RNA (siRNA). Assessment of knockdown with sexes pooled revealed ∼50% knockdown of Akt1/2 and ∼70% knockdown of Akt3 (top, left). Analyses by sex showed that siRNA directed against Akt1/2 knocked down Akt1/2 expression to an equivalent extent in both males and females (46 ± 3% vs. 43 ± 3% of control siRNA, respectively) without affecting expression of Akt3 (bottom, left). Similarly, expression of Akt3 was reduced to a comparable degree in both sexes by siRNA directed against Akt3 (50 ± 1 males vs. 53 ± 2 females, %control siRNA) with no change in expression of Akt1/2 (bottom, right). Immunoblot for Akt by sex is shown (top, right).

Fig. 6

Cardiomyocyte viability following Akt siRNA transfection. Knockdown of Akt1/2 eliminated ME-induced protection against hypoxia-reoxygenation cell death in males but only partially attenuated protection in females. Conversely, knockdown of Akt3 was ineffective in eliminating ME-induced protection in males but abrogated it in females. Control siRNA = green fluorescent protein (GFP). Shaded area indicates hypoxic period (0–90 min). Data are expressed as means ± SE.

Fig. 7

Akt Western blot analysis following Akt siRNA transfection. Isoform-specific Western blot analysis of p/t pan-Akt following stimulation with ME mirrors the viability data shown in Fig. 5. The data demonstrate robust phosphorylation in the presence of control siRNA (GFP sequence) in both males and females, only modest phosphorylation of Akt in males in the presence of Akt3 siRNA, and robust phosphorylation of Akt in females only in the presence of Akt1/2 siRNA. The data suggest that ME elicits p/t conversion mostly in Akt1/2 in males vs. mostly in Akt3 in females. Control siRNA = GFP. Data are expressed as means ± SE.