Estrogen dependence of sildenafil efficacy in female failing hearts (Gαq overexpressors). Gq-coupled receptor activation is a key contributor to various cardiac pathologies and a critical target for the cGMP signaling that underlies the antihypertrophy and remodeling effects of PDE5 inhibition. To examine the impact of estrogen levels on cardiac disease modulation induced by the PDE5 inhibitor sildenafil, mice with cardiac Gαq overexpression (Gq/oe), with or without surgical ovariectomy (OVX), were treated with sildenafil for 2 weeks (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI70731DS1). The Gαq overexpressor develops heart failure with slight hypertrophy as early as 3 to 4 weeks after birth (16). We found that OVX reduced the efficacy of sildenafil with regard to improving cardiac function as compared with controls (Figure 1, A and B). In non-OVX females, sildenafil ameliorated cardiac dysfunction more than in males (Supplemental Figure 2). Exogenous supplementation of estrogen (estradiol, E2) in OVX animals restored the efficacy of sildenafil (Figure 1, A and B, and Supplemental Figure 3A). The slight rise in cardiac hypertrophy in Gq/oe was itself normalized by E2 supplementation alone (Supplemental Figure 3B). A fetal gene marker of cardiac failure (BNP expression) as well as dysregulation of calcium-handling proteins (phospholamban phosphorylation and SERCA2a expression) (16, 17) were significantly improved by sildenafil in OVX mice with E2 rescue, but were unaffected in the absence of E2 (Supplemental Figure 3, C–E). Importantly, we found that OVX animals with E2 rescue as well as non-OVX animals showed very similar baseline characteristics and sildenafil responses, confirming that the appropriate dosage of exogenous E2 was used. These results suggest that the presence of estrogen critically impacts the response to PDE5 inhibition. We obtained similar results by more comprehensive analysis of cardiac function using pressure-volume (PV) analysis (Supplemental Figure 4). Sildenafil failed to increase myocardial PKG activity in OVX Gq/oe mice, but this activity was increased when E2 was exogenously provided (Figure 1C). This finding correlated with disparities in the repression of Gq signaling. We found that PKCα and calcineurin, both contributors to depressed cardiac performance (18) and maladaptive cardiac hypertrophy and remodeling (19), were markedly deactivated by sildenafil in OVX mice receiving E2, but were little impacted in OVX-only mice (Figure 1, D and E, and Supplemental Figure 3F).

Figure 1 Estrogen dependence of sildenafil efficacy in female failing hearts (Gq overexpressors). (A) FS echocardiographic changes before and after 2 weeks of vehicle or sildenafil treatment in wild-type and Gq-overexpressing (Gq/oe) female mice subjected to sham operation [non-OVX, OVX(–)], OVX [OVX(+)], or OVX with estrogen replacement [OVX(+) + E2] (n = 7–8 per group). *P < 0.05 versus the 2-week vehicle treatment group. (B) Summary data for FS increased after 2 weeks of sildenafil (SIL) treatment (n = 7–8 per group). *P < 0.05 versus non-OVX group; †P < 0.05 versus OVX group. (C) Myocardial PKG activity. (D) PKCα activity. (E) Calcineurin (Cn) protein expression was normalized to GAPDH (n = 4–7 per group). *P < 0.05 versus the wild-type groups in non-OVX or OVX mice; †P < 0.05 versus the vehicle-without-E2 groups in non-OVX or OVX Gq/oe mice; ‡P < 0.05 versus the other groups among OVX Gq/oe mice; §P < 0.05 versus the other groups among OVX mice. P values shown are for interactions between E2 and SIL treatments (E2 × SIL); 2-way ANOVA.

Estrogen dependence of sildenafil efficacy in female pressure-overloaded hearts and the essential role of PKGIα. We next extended this paradigm to a more general disease model using 2 weeks of pressure overload induced by transverse aortic constriction (TAC) (Supplemental Figure 5). We found that sildenafil had antihypertrophic/remodeling effects in OVX mice receiving E2, but it had no effect in OVX mice without E2 supplementation (Figure 2, A and B). Hypertrophy following TAC was reduced by E2 itself and by E2 combined with sildenafil (Figure 2C). We found that myocardial PKG activation was again dependent on the presence of E2 in OVX TAC mice (Figure 2D), and PKCα and calcineurin were deactivated by sildenafil only if E2 was supplemented (Supplemental Figure 6, A and B). The similarity of these data to those in the Gq/oe model supports the idea that estrogen plays a general role in PDE5-cGMP regulation in the stressed heart.

Figure 2 Estrogen dependence of sildenafil efficacy in female pressure-overloaded hearts and the essential role of PKGIα. Sildenafil response in OVX wild-type hearts exposed to 2 weeks of TAC with or without E2 replacement (A–D). (A) FS. (B) Myocardial BNP (Nppb) mRNA expression. (C) Heart weight (HW) normalized to tibia length (TL). (D) Myocardial PKG activity (n = 4–8 per group). Sildenafil response in OVX PKGIα-LZM hearts exposed to 2 weeks of TAC with or without E2 replacement (E–G). (E) FS. (F) Nppb mRNA expression. (G) HW/TL (n = 4–9 per group). *P < 0.05 versus the sham group; †P < 0.05 versus the vehicle-without-E2 group; ‡P < 0.05 versus the other groups in TAC; §P < 0.05 versus all other groups; P values shown are for interactions between E2 and SIL treatments; 2-way ANOVA.

To test whether this regulation was critically dependent on PKGIα, the predominant isoform in myocardium (20), TAC was used in mice with a knock-in mutation of PKGIα (Prkg1) harboring disrupted binding to leucine zipper–dependent interacting partners (PKGIα-leucine zipper mutant; PKGIα-LZM) (21, 22). Neither sildenafil, E2, nor their combination ameliorated the phenotype of PKGIα-LZM OVX hearts exposed to 2 weeks of pressure overload (Figure 2, E–G, and Supplemental Figure 6, C and D), indicating that changes in protein binding–dependent PKGIα signaling are required to achieve the beneficial effects of E2 and sildenafil.

cGMP production by estrogen coupled to the eNOS/sGC pathway as an upstream regulatory mechanism. We next identified the mechanism by which estrogen altered PKG activity responses to sildenafil. Supplementation with E2 did not alter myocardial PKGIα protein expression (Figure 3A) or PDE5 activity (Figure 3B), suggesting that the regulatory system downstream of cGMP was likely unaltered. However, cGMP synthesis was markedly stimulated in response to E2 in the presence of PDE5 inhibition in cardiac myocytes from OVX Gq/oe hearts (Figure 3C). This increase was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase (sGC) inhibitor, but not by the natriuretic peptide receptor A inhibitor HS142-1, suggesting functional coupling of E2 to NOS/sGC signaling. Consistent with this conclusion, we found that E2 supplementation in vivo failed to increase myocardial PKG activity in OVX hearts lacking eNOS (23) under pressure overload, while it increased PKG activity in OVX hearts lacking atrial natriuretic peptide (ANP) receptor (Npra–/–) (24), similar to what was observed in wild-type hearts (Figure 3D). These results indicate that estrogen/eNOS signaling in cardiac myocytes from female hearts provides tonic cGMP synthesis whose hydrolysis is targeted by PDE5.

Figure 3 cGMP production by estrogen coupled to the eNOS/sGC pathway as an upstream regulatory mechanism. (A) PKGIα protein expression normalized to GAPDH. (B) PDE5 activity in Gq/oe female hearts subjected to sham operation (non-OVX), OVX, and OVX with E2 replacement (n = 5–8 per group). (C) Cardiac myocyte cGMP synthesis in OVX Gq/oe hearts. Isolated cardiac myocytes were treated with E2 (1 nM), E2 plus 10 μM ODQ sGC inhibitor, E2 plus 100 μg/ml HS142-1 (ANP receptor inhibitor), ODQ alone, or HS142-1 alone (n = 3–4 per group). (D) Myocardial PKG activity in OVX hearts lacking eNOS (n = 5–6 per group) or lacking NPRA (n = 4–5 per group) exposed to 2 weeks of TAC, with or without E2 replacement, as well as wild-type controls. *P < 0.05; P values shown are for interactions between E2 and TAC; 2-way ANOVA.

Gender differences in sildenafil responses and the mechanism involving eNOS-cGMP synthesis and PKG activation under cardiac stress. Our previous work demonstrated that sildenafil ameliorated TAC-induced hypertrophic remodeling in male hearts (5–7), which have low levels of estrogen. Therefore, we tested whether male hearts were equipped with a PDE5-coupled cGMP regulatory system that is less estrogen dependent. In isolated male Gq/oe cardiac myocytes, E2 stimulated cGMP synthesis via the eNOS/sGC pathway (Figure 4A), as was observed in the female counterparts. Administration of E2 to male Gq/oe hearts in vivo provided additive benefits to sildenafil treatment in terms of cardiac function (percentage of fractional shortening [FS]), repression of BNP, and deactivation of Gq-related signaling (Rcan1 and PKCα), while sildenafil alone showed significant effects (Figure 4B and Supplemental Figure 7, A–D). Unlike Gq/oe OVX females, male Gq/oe myocardium had a resting increase in PKG activity compared with that seen in controls, and this was further increased by E2 administration as well as by sildenafil in an additive (no interaction) manner (Figure 4C). These changes correlated with molecular profiles (Supplemental Figure 7, B–D). The slight rise in cardiac hypertrophy in male Gq/oe hearts was again normalized by E2 administration alone (Supplemental Figure 7E). We observed similar additive beneficial effects of E2 and sildenafil treatments in male pressure-overload models (Supplemental Figure 8). Thus, the primary difference in sildenafil efficacy between OVX females and males derives from the drug’s dependence on estrogen, as the estrogen levels themselves are comparable (25). We further examined this difference in isolated cardiomyocytes in acute settings. Male Gq/oe myocytes had no resting increase in PKG activity compared with wild-type control myocytes (likely reflecting the loss of neurohormonal stimulation with cell isolation) and compared with OVX female Gq/oe myocytes, and only male cells displayed a rise in this activity with sildenafil (Figure 4D). Estrogen augmented PKG activity in myocytes from both sexes, and in the presence of E2, both displayed further increases in this activity with sildenafil. These results support the rapid nongenomic action of estrogen in this regulation and also indicate that estrogen-independent cGMP synthesis induction exists in male Gq/oe cardiac myocytes, as PDE5 activity and PKGIα expression in male hearts were comparable to what we observed in female hearts (data not shown). In all, we examined the activation status of eNOS in Gq/oe hearts in male mice and in female mice with or without OVX. Importantly, in male hearts, Gq/oe itself induced a significant increase in eNOS phosphorylation at serine 1177, whereas the presence or absence of OVX, but not of Gq/oe, determined the phosphorylation in female hearts (Figure 4E). We found that myocardial NOS activity corresponded with the status of eNOS phosphorylation (Figure 4F). These results demonstrate the significant difference in PDE5-coupled cGMP regulation between the sexes: the eNOS-cGMP synthetic machinery is stress-responsive in male hearts and is tonic and estrogen dependent in female hearts.