A novel assay to assess the effects of estrogen on the cardiac calmodulin binding equilibrium

The Ca2+-binding protein calmodulin (CaM) modulates numerous target proteins but is produced insufficiently to bind all of them, generating a limiting CaM equilibrium. Menopause increases cardiac morbidity; however, it is unknown if the cardiac CaM equilibrium is affected by estrogen. We devised an assay to assess the effects of ovariectomy and estrogen treatment on the cardiac CaM equilibrium. Materials and

Sprague-Dawley rats received sham surgery or ovariectomy, followed by 2-week treatment with vehicle or 17β-estradiol. Ca2+-saturated left ventricular (LV) lysates were processed through CaM sepharose columns, which retained CaM-binding proteins unoccupied by endogenous CaM. Eluants therefrom were subjected to a competitive binding assay against purified CaM and a CaM biosensor to assess the amounts of unoccupied CaM-binding sites. LV cellular composition was assessed by immunohistochemistry. Key findings: LV eluants processed from sham animals reduce biosensor response by ~32%, indicating baseline presence of unoccupied CaM-binding sites and a limiting CaM equilibrium. Ovariectomy exacerbates the limiting CaM equilibrium, reducing biosensor response by ~65%. 17β-estradiol treatment equalizes the difference between sham and ovariectomized animals. These changes reflect whole tissue responses and are not mirrored by changes in total surface areas of cardiomyocytes and fibroblasts. Consistently, Ca2+-dependent, but not Ca2+-independent, interaction between CaM and the cardiac inositol trisphosphate receptor (IP3R) is reduced following ovariectomy and is restored by subsequent 17β-estradiol treatment. Significance: Our assay provides a new parameter to assess tissue CaM equilibrium. The exacerbated limiting CaM equilibrium following estrogen loss may contribute to cardiac morbidity and is prevented by estrogen treatment.

Our assay provides a new parameter to assess tissue CaM equilibrium. The exacerbated limiting CaM equilibrium following estrogen loss may contribute to cardiac morbidity and is prevented by estrogen treatment.