Nuclear translocation of the cardiac L-type calcium channel C-terminus is regulated by sex and 17β-estradiol.
The cardiac voltage gated l-type Ca(2+) channel (Cav1.2) constitutes the main entrance gate for Ca(2+) that triggers cardiac contraction. Several studies showed that the distal C-terminus fragment of Cav1.2 α1C subunit (α1C-dCT) is proteolytically cleaved and shuttles between the plasma membrane and the nucleus, which is regulated both developmentally and by Ca(2+). However, the effects of sex and sex hormone 17β-estradiol (E2, estrogen) on α1C-dCT nuclear translocation are still unexplored. To investigate the sexual disparity in the α1C-dCT nuclear translocation, we first generated an antibody directed against a synthetic peptide (GRRASFHLE) located in α1C-dCT, and used it to probe ventricular myocytes from adult female and male mice. Immunocytochemistry of isolated mouse primary adult ventricular myocytes revealed both nuclear staining and cytosolic punctuate staining around the T-tubules. The ratio of nuclear to cytosolic intensity (Inuc/Icyt) was significantly higher in isolated female cardiomyocytes (1.42±0.05) compared to male cardiomyocytes (1.05±0.02). Western blot analysis of nuclear fraction confirmed these data. Furthermore, we found a significant decrease in nuclear staining intensity of α1C-dCT in both female and male cardiomyocytes upon serum withdrawal for 18h (Inuc/Icyt 1.05±0.02 and 0.89±0.02, respectively). Interestingly, subsequent E2 treatment (10(-8)M) for 8h normalized the intracellular distribution of α1C-dCT in male cardiomyocytes (Inuc/Icyt 1.04±0.02), but not in female cardiomyocytes. Acute treatment of male cardiomyocytes with E2 for 45min revealed a similar effect. This effect of E2 was revised by ICI indicating the involvement of ER in this signaling pathway. Taken together, our results showed that the shuttling of α1C-CT in cardiomyocytes is regulated in a sex-dependent manner, and E2-activated ER may play a role in the nuclear shuttling of α1C-dCT in male cardiomyocytes. This may explain, at least partly, the observed sex differences in the regulation of cardiac Cav1.2 channel activity.
Max-Delbrueck-Center for Molecular Medicine in the Helmholtz Association, Molecular Muscle Physiology, Berlin, Germany; DZHK (German Center for Cardiovascular Research), Partner Site Berlin, Berlin, Germany. Electronic address: shokoufeh.mahmoodzadeh@mdc-berlin.de.
S
S
Mahmoodzadeh
Max-Delbrueck-Center for Molecular Medicine in the Helmholtz Association, Molecular Muscle Physiology, Berlin, Germany.
H
H
Haase
Maxe-Delbrueck-Center for Molecular Medicine in the Helmholtz Association, Advanced Light Microscopy, Berlin, Germany.
A
A
Sporbert
Max-Delbrueck-Center for Molecular Medicine in the Helmholtz Association, Electrochemical Signaling in Development and Disease, Berlin, Germany.
T
T
Rharass
DZHK (German Center for Cardiovascular Research), Partner Site Berlin, Berlin, Germany; Max-Delbrueck-Center for Molecular Medicine in the Helmholtz Association, Electrochemical Signaling in Development and Disease, Berlin, Germany.
D
D
Panáková
Max-Delbrueck-Center for Molecular Medicine in the Helmholtz Association, Molecular Muscle Physiology, Berlin, Germany; DZHK (German Center for Cardiovascular Research), Partner Site Berlin, Berlin, Germany.
I
I
Morano
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D006352:Heart Ventricles / Q000378:metabolism; D008297:Male; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D012737:Sex Factors
D020746:Calcium Channels, L-Type; D004958:Estradiol
17β-estradiol; Ca(2+) channel; Cardiomyocyte; Nuclear translocation