Previous studies have shown that estrogen exerts myocardial protective effects in models of pressure overload–induced cardiac hypertrophy5
and myocardial infarction.21
However, the effect of estrogen in preventing the development of CHF has not been investigated. In the present study, we extend the effects of estrogen to the development of CHF in a genetic model of dilated cardiomyopathy. The Gq transgenic mice exhibit differing phenotypes depending on the number of copies of the Gq transgene. For example, with 4 copies of the Gq transgene, mutant Gq mice develop progressive cardiac hypertrophy without left ventricular dilatation or CHF.17
However, with increased Gq transgene copy number, such as the mice used in the present study that had 40 copies of the transgene, the mutant male Gq mice spontaneously develop mild cardiac hypertrophy, with rapid progression to dilated cardiomyopathy. Using these male Gq transgenic mice, we found that treatment with estrogen attenuated the development of CHF through antioxidative and antiapoptotic mechanisms. Specifically, we found that estrogen decreased myocardial oxidative stress by inhibiting NADPH oxidase activity, reducing ROS generation, and increasing TrxRD activity in the hearts of Gq mice. The antiapoptotic effect of estrogen was blocked by an inhibitor of TrxRD, azelaic acid, which suggests that TrxRD is an important mediator of cardiomyocyte survival in CHF. These findings suggest that estrogen therapy may have benefits in patients with CHF through upregulation of TrxRD. However, to determine the relative contribution of TrxRD to the protective effects of estrogen on CHF, further studies will need to be performed with estrogen in mice lacking TrxRD.
Myocardial oxidative stress contributes to the rapid and progressive deterioration of cardiac function in patients with CHF.7
For example, NADPH oxidase–linked ROS activity has been found to be elevated in failing hearts of patients with either ischemic or dilated cardiomyopathy.6
Indeed, increased oxidative stress from pressure overload or Ang II is associated with increased cardiac remodeling and fibrosis,22,23
and antioxidant therapy has been shown to decrease cardiac fibrosis.24
The present results are consistent with these findings, because NADPH oxidase activity and ROS generation were both increased in the hearts of Gq transgenic mice. Treatment with estrogen reduced NADPH oxidase activity and ROS production and attenuated the severity of CHF in Gq transgenic mice. These findings are similar to previous studies showing that estrogen attenuates the development of cardiac hypertrophy by inhibiting the expression of NADPH oxidase25
and downregulating Rac1 GTPase.26
In addition, estrogen inhibits cardiac hypertrophy by decreasing ROS production not only through inhibition of NADPH oxidase activity but also by increasing antioxidants, such as superoxide dismutase.27
Thus, the findings of previous studies with regard to estrogen and cardiac hypertrophy are somewhat similar to the present findings with estrogen and CHF. A consequence of increased oxidative stress is that ROS may trigger apoptotic cell death.28
Because increased cardiomyocyte apoptosis is an important contributor to the loss of cardiomyocytes after maladaptive hypertrophy29
and heart failure,30
factors that regulate cellular apoptosis may affect cardiac size and contractility in CHF. Estrogen has been shown to inhibit cellular apoptosis in cardiomyocytes.31
In the present study, this was associated with estrogen’s ability to prevent the development of CHF in Gq transgenic mice. Although decreasing myocardial oxidative stress could contribute to the antiapoptotic effect of estrogen, we also found that estrogen increased the activity of protein kinase Akt in the hearts of Gq transgenic mice. The phosphatidylinositol 3-kinase/Akt pathway prevents cellular apoptosis by activating downstream cell-survival pathways.32
Indeed, activation of Akt in the heart by estrogen has also been shown to prevent cardiomyocyte apoptosis.21
The Trx system is an important cellular antioxidant defense mechanism. In mice with cardiac-specific overexpression of a dominant-negative mutant of the Trx1 gene, oxidative stress is increased in the pressure-overloaded hypertrophic heart.33
In contrast, overexpression of Trx in transgenic mice attenuates doxorubicin-induced cardiotoxicity by reducing myocardial oxidative stress.34
In a similar fashion, we found that upregulation of the Trx system by estrogen inhibited myocardial oxidative stress and attenuated the severity of CHF in Gq transgenic mice. Indeed, Trx and TrxRD are upregulated by estrogen in a variety of other tissues.15,16
Interestingly, we showed that E2 upregulates the cardiac Trx system in several different cellular compartments (ie, cytoplasm and mitochondria), which suggests that Trx may mediate multiple effects of estrogen on the heart. It remains to be determined by what mechanism the estrogen regulates Trx and TrxRD expression, given that neither the Trx nor the TrxRD promoter contains a canonical estrogen response element; however, these gene promoters contain half-estrogen response elements (GGTCA) and several SP1 sites, which have been shown to synergize with the estrogen receptor on various promoters.35
Thus, it would be genomic effects that induced Trx and TrxRD expression by estrogen. More detailed promoter studies will be required to examine these mechanisms.
Trx interacts with ASK1 through its direct binding to the N-terminal noncatalytic region of ASK1 and regulates ASK1 activity through Trx.36
ASK1 is inactivated by binding with Trx-(SH)2
, which leads to ubiquitination and degradation of ASK1. Conversely, oxidization of Trx results in dimerization and activation of ASK1.37
In the present study, upregulation of TrxRD activity by estrogen increased Trx-(SH)2
and reduced ASK1 activity. ASK1 is a member of the MAPK kinase kinase that activated the JNK and p38 MAPK pathways. 38
Prolonged activation of JNK and p38 MAPK may aggravate the pathological changes in the heart through the proapoptotic action of these MAPKs. Therefore, inhibition of ASK1 activity and its downstream targets, JNK and p38 MAPK, by estrogen may contribute to some of the antiapoptotic effects of estrogen in CHF.
In the present study, we used a high dose of estrogen that maintains E2 serum concentrations 20-fold higher than the physiological range.39
Female Gq mice have been reported to exhibit a particularly severe phenotype of peripartum cardiomyopathy with the first pregnancy,40
so the physiological range of estrogen may have no cardioprotective effect in this Gq mouse model. Accordingly, we chose high-dose E2 treatment. High-dose E2-treated Gq mice fail to gain weight. This might reduce the susceptibility to heart failure in Gq mice and lead to a beneficial effect on the development of CHF. Recently, Beer et al41
reported that high-dose E2 treatment almost completely prevents development of post–myocardial infraction remodeling. However, the use of high-dose E2 is limited by adverse side effects such as an increased risk of thrombosis and promotion of breast and endometrial cancer and, in males, by a feminizing effect. Thus, at present, long-term treatment with high-dose E2 is not feasible for prevention of CHF in humans. Further studies will be needed to address the effects of estrogen-related compounds such as selective estrogen receptor modulators. Alternatively, targeting TrxRD or ASK1 may be a more feasible approach than estrogen therapy in terms of preventing CHF.
In summary, estrogen improves cardiac contractility and prevents progressive cardiac enlargement in a genetic mouse model of CHF by activating TrxRD, inhibiting NADPH oxidase activity, and reducing oxidative stress in the heart. The upregulation of the Trx system by estrogen leads to inhibition of ASK1-mediated cardiomyocyte apoptosis. Although we have demonstrated that estrogen is beneficial in a mouse model of CHF, it remains to be determined whether estrogens are useful adjunctive therapy in patients with CHF. Further clinical trials with estrogen therapy in heart failure are needed to address this issue.