Chronic increased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17β-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55 ± 4.80 vs. E2: 6.51 ± 3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13 ± 3.66 vs. E2: 2.18 ± 1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.

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Estrogen (17β-estradiol, E2) is one of the key regulators of growth, differentiation and physiological function in different tissues, including the heart. The effects of E2 are mainly mediated by estrogen receptor (ER) alpha and beta, which act in concert with many cofactors to mediate estrogenic effects. So far, only a few cofactors of ER have been described in the human heart. To gain a better understanding of E2-mediated ERα action in the human heart, we identified and characterized the novel interaction partners of ERα Yeast two hybrid screening of a human heart cDNA library revealed that ERα interacts with the cardiac myosin essential light chain isoform 4 (ALC1) in presence of E2. ALC1, as a member of contractile proteins, is expressed in the fetal heart and becomes restricted to the atria of the adult heart under physiological conditions, and is reexpressed in ventricle of adult hypertrophic hearts. This switch is accompanied by alteration of contractile performance, thus improving the heart function. Retransformation experiments showed that ALC1 interacts with full-length ERα and ERα-EF domain in presence of E2. The interaction of ERα with ALC1 was also confirmed by Co-IP in human atrium. Double irnmunofluorescence (IF) analysis of paraffin-embedded sections from human atrium tissues showed co-localization of ERα and ALC1 proteins in a striated sarcomeric pattern. Co-localization corresponds most likely to the H-zone of sarcomere. IF analysis of AC16 cells (a human cardiomyocytes cell line) showed that ERα and ALC1 were mainly localized in the cytoplasm in the absence of E2. However, E2 treatment of AC16 cells led to a translocation of ALC1 and ERα into the nuclei of AC16 cells, where they also co-localized. Expression analysis in AC16 cells showed that E2 increases the expression of ALC1 gene (MYL4). Using ERE-based luciferase reporter assays we showed that E2-induced interaction of ERα with the ALC1 represses the transcriptional activity of ERα. We characterize for the first time an E2-regulated interaction of ALC1 with ERα in cardiomyocytes that may be crucial in physiological and/or pathological processes by regulating of transcriptional activity of ERa in the heart and/or by modulating contractile properties of cardiomyocytes.