Sex differences in cardiomyocyte connexin43 expression

Abstract

Decreases in cardiac connexin43 (Cx43) play a critical role in abnormal cell-to-cell communication and have been linked to the resistance of the female heart to arrhythmias. We therefore hypothesized that Cx43 expression would be greater in female cardiomyocytes than in male cardiomyocytes under pathologic conditions. Adult ventricular myocytes were isolated from male and female rats and treated with phenylephrine (PE), a well-established pathologic stimulus. Cx43 gene and protein expression was determined. The expression of micro-RNA-1 (miR-1), a micro-RNA known to control Cx43 protein expression in cardiomyocytes, was also determined. Cx43 mRNA and protein levels were significantly higher in the female cardiomyocytes than in the male cardiomyocytes (mRNA: 1.4-fold; Protein: 5-fold, both P < 0.05) under both basal and pathologic conditions. PE treatment increased Cx43 expression only in female cardiomyocytes. Cx43 phosphorylation, a marker of preserved Cx43 function, was also higher (P < 0.05), and The expression of miR-1 was lower (P < 0.05) in the female cardiomyocytes after PE treatment. The expression of miR-1 was unchanged by PE treatment in male cardiomyocytes. Thus, a sex difference in miR-1 may be responsible for the sex difference in Cx43 expression in cardiomyocytes under pathologic conditions. Taken together, our results demonstrate a sex difference in Cx43 expression and site-specific phosphorylation that favors cardioprotection in female cardiomyocytes.

Figure 1

Pathologic gene induction by phenylephrine (PE) in ARVMs. Male and female ARVMs were isolated as outlined in materials and method and treated for 24 hours with PE (10 mcM). Brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and β-myosin heavy chain isoform (β-MyHC) mRNA expression were determined by quantitative RT-PCR. PE treatment produced similar induction of BNP (A), ANP (B) and β-MyHC (C) in both male and female ARVMs indicated a similar pathologic response between the sexes. 20 hearts/group, n = 30/group. Control vs PE: * P<0.05, ** P<0.01, *** P<0.0001.

Figure 2

Connexin 43 (Cx43) expression under basal and pathologic conditions. Female ARVMs demonstrate higher Cx43 mRNA (Panel A) and protein (Panels B–F) expression than male ARVMs under control and PE treated conditions. PE treatment increases Cx43 mRNA expression in both male and female cardiomyocytes (Panel A). C: Representative Western Blot of Cx43 phosphorylation isoforms separated by SDS-PAGE under control (C), PE and PE+Prazosin (PPZ) treated conditions. All protein data is normalized to Calnexin, used as a loading control. The faster migrating form includes the non-phosphorylated isoform (P0). There are also at least 2 slower migrating isoforms (P1 and P2). Although PE treatment increases the P1 isoform in male ARVMs (Panel E), PE treatment increases total Cx43 and the P1 and P2 isoforms female ARVMs (Panels C, E, F). mRNA: 20 hearts/group, n = 30/group; Protein: 11 hearts/group. Control vs PE: * P<0.05, ** P<0.01; Male vs Female: † P<0.05, †† P<0.005, ††† P<0.0005.

Figure 3

Densitometric analysis (Panel A) and representative Western Blots (Panel B) of expression of the Connexin 43 (Cx43) isoform phosphorylated at serine 368 (S368) under basal and pathologic conditions. Female ARVMs demonstrate higher phosphorylation at S368 (p368) under both basal and PE treated conditions. Phosphorylated S368 isoforms are higher following PE treatment in both male and female ARVMs. Panel C: Representative Western blot of nonphosphorylated Cx43 at S368 (np368). 4 hearts/group, n = 8/group. Control vs PE: * P<0.05, ** P<0.01; Male vs Female: † P<0.05, †† P<0.01.

Figure 4

microRNA-1 (miR-1) expression is lower in female but not male ARVMs following PE treatment. A decrease in miR-1 expression removes repression of Cx43 protein expression in the female ARVMs. 9 hearts/group, n = 13/group. Control vs PE: * P<0.05.