Cardiac PAD2 expression and myocardial citrullination decline with age in female mice independent of estrogen.

Cardiac aging is sexually dimorphic, with women more likely than men to develop diastolic dysfunction for which no therapies exist. Loss of estrogen (E2) during menopause increases the risk of diastolic dysfunction in women through unclear mechanisms. Citrullination, a posttranslational modification catalyzed by peptidylarginine deiminase 2 (PAD2), is positively regulated by E2, suggesting a novel mechanism linking PAD2, diastolic function, and E2 in the female heart. We hypothesized that PAD2 expression and citrullination are sexually dimorphic with aging such that as E2 levels decline, so does PAD2 expression and citrullination, contributing to diastolic dysfunction. PAD2 expression decreased with age in female mice but not in the aging male heart. Mass spectrometry detected citrullination of sarcomeric and metabolic proteins, with lower levels of citrullinated proteins in aged female hearts compared with young. To confirm direct regulation of PAD2 by E2, a cohort of young (2 mo) and aged (21 mo) mice underwent ovariectomy with or without E2 replacement. Contrary to our hypothesis, PAD2 expression was not regulated by E2 in the heart. To directly link PAD2 and diastolic function, we assessed cardiac function in middle-aged female global PAD2 knockout mice and found that loss of PAD2 resulted in diastolic dysfunction. Together, we establish that protein citrullination and PAD2 decline with age in the female heart, perhaps contributing to diastolic dysfunction. Elucidation of the mechanisms underlying PAD2 declines in the female heart remains to be determined and may benefit the development of therapies for diastolic dysfunction for aging women.