Abstract

   Estrogen exerts diverse biological effects in multiple tissues in both
   animals and humans. Much of the accumulated knowledge on the role of
   estrogen receptor (ER) in the heart has been obtained from studies
   using ovariectomized mice, whole body ER gene knock-out animal models,
   ex vivo heart studies, or from isolated cardiac myocytes. In light of
   the wide systemic influence of ER signaling in regulating a host of
   biological functions in multiple tissues, it is difficult to infer the
   direct role of ER on the heart. Therefore, we developed a mouse model
   with a cardiomyocyte-specific deletion of the ERα allele (cs-ERα^−/−).
   Male and female cs-ERα^−/− mice with age/sex-matched wild type controls
   were examined for differences in cardiac structure and function by
   echocardiogram and differential gene expression microarray analysis.
   Our study revealed sex-differences in structural parameters in the
   hearts of cs-ERα^−/− mice, with minimal functional differences.
   Analysis of microarray data revealed differential variations in the
   expression of 208 genes affecting multiple transcriptional networks.
   Furthermore, we report sex-specific differences in the expression of 56
   genes. Overall, we developed a mouse model with cardiac-specific
   deletion of ERα to characterize the role of ERα in the heart
   independent of systemic effects. Our results suggest that ERα is
   involved in controlling the expression of diverse genes and networks in
   the cardiomyocyte in a sex-dependent manner.

Introduction

   Estrogen receptors (ER) are involved in multiple biological processes
   in a variety of tissues including the cardiovascular system, adipose
   tissue, and sex organs [37][1]. Generally, the actions of ERs are
   triggered upon binding of estrogens such as 17-β estradiol (E2), the
   predominant active form of estrogen [38][2]. Upon ligand binding, ERs
   mediate their response by two possible modes of action: genomic, which
   elicits a slow response, and non-genomic, which induces a rapid
   response. Genomic activation of ERs initiate transcription factors
   either by direct DNA interaction through estrogen response elements
   (ERE) or through ER-DNA indirect interactions by tethering with known
   transcription factors bound to the DNA [39][3]. The non-genomic
   actions, on the other hand, are mostly membrane-initiated and involve
   signaling cascades, such as the mitogen-activated protein kinase (MAPK)
   pathway, the cyclic adenosine mono-phosphate/protein kinase A
   (cAMP/PKA) pathway, or the endothelial nitric oxide synthase (eNOS)
   pathway [40][4]–[41][6].

   ERs have been implicated in several pathophysiological conditions
   including diabetes and obesity with implications for cardiovascular
   disease [42][1], [43][7]. In the heart, cardiomyocytes express both
   sub-types of ER, ERα and ERβ, with significantly higher levels of ERα
   [44][8]–[45][10]. Mice with whole-body deletion of ERα have been shown
   to exhibit altered cardiac substrate preference, particularly uptake
   and maintenance of glucose in the heart [46][11]. Indeed, whole-body
   ERα knockout mice are obese and insulin resistant [47][12]–[48][14] and
   exhibit diminished rates of fatty acid (FA) oxidation in skeletal
   muscle [49][13]. ERα is also thought to possess cardio-protective
   properties, due to its ability to up-regulate expression of ApoE, an
   apolipoprotein that increases clearance of low density lipoproteins
   (LDL) from circulation [50][15], [51][16]. Similarly, ERβ has been
   reported to mediate sex-differences in ischemia/reperfusion injury
   [52][17], [53][18] as well as protect against left-ventricular
   hypertrophy (LVH) in females [54][19]. Interestingly, ERα and ERβ have
   also been reported to differentially modulate the expression of
   inflammatory markers, in particular that of inducible nitric oxide
   synthase (iNOS) [55][20]. Nevertheless, to date, the studies describing
   the role of ERs on the cardiovascular system were based on whole-body
   ER knockouts or on the effects of ER ligands, such as estrogen, on the
   heart, but with the caveat that such ER ligands also affect peripheral
   tissues. In light of the wide influence of ER signaling in regulating
   physiologic functions in multiple tissues, including systemic energy
   homeostasis, and considering that peripheral substrates can induce
   cardio-metabolic remodeling, it is difficult to infer the direct role
   of ER on the heart using whole-body KO models.

   As a first step in characterizing the role of ERs in the heart,
   independent of systemic effects of ERs, we have generated a mouse model
   with a cardiomyocyte-specific deletion of ERα (cs- ERα^−/−. The
   availability of mice with cs-ERα^−/− will enable, for the first time,
   to investigate the role of ERα in cardiac tissue independent of
   peripheral effects. In addition, since ERs are important therapeutic
   targets, development of in vivo models of selective modulation (such as
   cardiac specific deletion) will enable better understanding of ER
   effects in specific tissue [56][21], [57][22]. To that end, we
   characterized basal cardiac structure and function, and performed gene
   expression microarray profiling to determine differentially affected
   networks and pathways in male and female mice. Statistically validated
   results from the microarray analyses were used for pathway analysis,
   with emphasis on transcriptional factors and receptor networks (for
   deciphering genomic actions of ERα). Our findings indicate that cs-
   ERα^−/− manifests significant variations in the expression profile of
   genes involved in metabolism, cell growth and differentiation, muscle
   architecture, and relaxation. Finally, we delineate sex differences
   associated with the absence of ERα in the heart and identify key
   transcriptional/receptor hubs that are involved in ERα mediated
   regulation/signaling in cardiac tissue.

Materials and Methods

   All experiments were conducted according to a protocol approved by the
   animal experiment committee at Washington University School of Medicine
   in Saint Louis (IACUC Animal Welfare Assurance # A-3381-01) and in
   accordance to ‘Principles of laboratory animal care’ (NIH pub no.
   85–23, revised 1985;
   [58]http://grants1.nih.gov/grants/olaw/references/phspol.htm). Animals