Is a Decline in Myocardial Citrullination in the Aged Female Heart Really PAD 2 the Bone?

Sexual dimorphism exists in many biological processes that directly and indirectly impact cardiac physiology, and many common cardiovascular diseases exhibit sex-dependent differences^1,2. In fact, heart failure with reduced ejection fraction (HFrEF), or systolic dysfunction, is more prevalent in men, and heart failure with preserved ejection fraction (HFpEF), or diastolic dysfunction, is more prevalent in aged women^1,2. Diastolic dysfunction is characterized by impaired ventricular relaxation, increased myocardial pressures, and decreased ventricular filling, all of which culminate in reduced ventricular compliance. Despite many proposed underlying mechanisms, sarcomeric dysfunction is a significant contributor to diastolic dysfunction. Indeed, both mutations and post-translational modifications in many sarcomeric proteins have been shown to impact the Ca²⁺ sensitivity of the contractile myofilaments, ultimately contributing to diastolic dysfunction. Although advanced age and hormonal status have been shown to be significant risk factors for the development of diastolic dysfunction in women^1,2, little is known regarding the mechanisms that promote diastolic dysfunction in the aged female heart and the sex-dependent differences in myocardial stress responses. In addition, few therapeutics are available to target diastolic dysfunction, creating a desperate need to understand potential regulators in greater detail.

In recent years, the irreversible post-translational modification, citrullination, has garnered attention. Citrullination is the conversion of a negatively charged arginine to a neutrally charged citrulline, which is catalyzed by the peptidylarginine deaminase (PAD) family of enzymes. The PAD family of enzymes comprises PAD1, PAD2, PAD3, PAD4, and PAD6, with PAD2 and PAD4 being predominantly expressed in the heart. Specifically, studies have shown that PAD2 is expressed in cardiomyocytes and fibroblasts³, and PAD4 is expressed in several immune cell populations, including neutrophils and macrophages³. It has been documented that with cardiac disease, PAD2 expression increases, resulting in the aberrant citrullination of several proteins^3,4. In addition, PAD2 inhibition has been shown to protect from disease³. However, to date, the extent to which PAD2-mediated citrullination contributes to diastolic dysfunction and whether PAD2-mediated citrullination of cardiac proteins plays a key role in age- and sex-dependent changes in cardiovascular physiology remain unknown, which were both goals of the present study by Shorthill et al⁵.

The study by Shorthill et al⁵., provides emerging insights into age- and sex-dependent changes in PAD2 expression and the citrullination of cardiac proteins. In fact, before this study, the contributions of PADs and citrullination to sex-specific differences in the cardiovascular system had not been explored, with many studies using predominantly male animals. The authors found that PAD2 expression was reduced in aged female hearts when compared to young females and both young and aged males⁵. However, given that cardiac levels of PAD2 were examined at both 2 and 20 months in male and female mice, it remains unclear when PAD2 levels begin to decline in female mice. Diastolic dysfunction was also observed in female mice with global PAD2^−/− at 14 months of age compared to wild-type females; however, whether young PAD2 mice of both sexes exhibit diastolic dysfunction independent of aging remains undetermined. Knowing when PAD2 levels decline in female hearts will not only shed more light on what drives the decrease but could also help determine whether this decline occurs before or as a result of the onset of age-dependent diastolic dysfunction. Of note, in the present study, PAD2 levels in the aged female heart did not correlate with a significant reduction in overall citrullination levels. Although this could be a result of the smaller sample size used, it may suggest that PAD2-independent citrullination is occurring, and its contribution should be examined in future investigations.

The study by Shorthill and colleagues⁵ identified several citrullinated proteins in the hearts of male and female animals (highlighted in Fig. 1). This corroborated some of the previously identified citrullinated proteins in the heart⁴. Notably, the present study is the first to investigate sex-dependent changes in cardiac protein citrullination. The present studies highlighted sexual dimorphism in the citrullination of tropomyosin, whereby a reduction was observed in aged female hearts that was absent in male hearts⁵. This is particularly interesting, given the importance of tropomyosin in maintaining diastolic function, and could be a key factor contributing to sex-dependent differences in cardiac function with age. Despite this, the physiological impact of this change is unclear. Additional protein changes may contribute to diastolic dysfunction in aged wild-type female mice; however, whether these same proteins are implicated in the PAD2^−/− model remains unclear. Given the large number of citrullinated mitochondrial and sarcomeric proteins identified in the male and female hearts examined, it seems likely that significant changes in metabolism may also contribute, in addition to changes at the level of the sarcomere. Additional mechanistic investigation of the physiological consequences of citrullination on the respective functions of these proteins could hold the key to uncovering new insights into sex-specific changes in cardiac function. Whether these citrullinated proteins are also modified by additional post-translational modifications, such as phosphorylation, oxidation, and S-nitrosylation⁶, in an age- and sex-dependent manner, would be of interest to understand. This is especially important given the known crosstalk between different post-translational modifications, which could further shape age- and sex-dependent responses in cardiac function.

Figure 1. Potential Contributions of PAD2 in Myocardial Aging and Future Insights.

This figure summarizes the key findings of the work conducted by Shorthill et al., and the key citrullinated proteins (notated by gene name) identified in male and female hearts. It also provides key areas for future investigations to fully elucidate the role of PAD2 and citrullination in sex-dependent changes in the aged myocardium. Abbreviations: 17β-estradiol, E2; Peptidyl arginine deiminase 2, PAD2. (Created in BioRender. Collins, H. (2025) https://BioRender.com/8eigs8b).

The study by Shorthill et al.⁵ also showed that, unlike uterine tissues, where PAD2 expression is regulated by 17β-estradiol (E2), this was not the case in the heart, because E2 and E2 depletion had minimal impact on changes in PAD2 expression. Given that PAD2 may function as an estrogen-receptor coactivator, it is necessary to determine receptor activation and expression in this context to see if PAD2 may be functioning differently in the heart. In addition to E2, several factors have been proposed to regulate the expression and activity of PAD2, including the transcription factors Sp1 and Sp3⁷, regulation of its expression through citrullination of histone H3, autocitrullination⁸, and fluctuations in Ca²⁺ levels³, amongst others. Although the authors⁵ suggest this could be a consequence of the long 3’ UTR during the translation of PAD2, the data suggest the decrease could also be the result of modifications to histone H3 citrullination. Regardless, it will be essential to examine what is contributing to this decline in cardiac PAD2 in greater detail to gain further insights into what may drive changes in PAD2 expression between the aged sexes.

Although the present study potentially rules out a role for E2 in PAD2-mediated citrullination, it is important to note that with aging and the onset and progression of menopause, the contributions of estrogens change. Specifically, the estrogen, estrone, has been shown to increase during menopause⁹, which has not been examined in the present study. In fact, many studies of estrogen replacement in mouse ovariectomy (OVX) models often use E2 to rule in or out the contributions of hormones to sex-dependent findings. Understanding the role of different estrogens in the context of cardiovascular studies will provide a deeper understanding of their specific contributions to age-dependent changes. In addition, as the authors clearly note, the OVX model impacts other hormones, including progesterone; therefore, using a model that does not affect these other hormones, replacing these hormones, or using one that has a gradual hormonal decline, such as the VCD model of menopause¹⁰, could be necessary to fully understand the regulation of PAD2 in the heart. Furthermore, given the noted regulation of PAD2 by testosterone by the authors⁵, it would be interesting to determine whether gonadectomy in males results in similar PAD2-mediated changes as those observed in aged females.

Given the phenotypical differences between mice with age-related decreases in PAD2 and the global PAD2^−/− model, other potential mechanisms could be at play. Indeed, alterations in extracellular matrix composition and immune cell responses, which occur in the aging heart, can contribute to diastolic dysfunction¹ and have been previously shown to differ between sexes². Because PAD2 is expressed in both cardiac fibroblasts and cardiomyocytes, the specific contribution of the two cell populations to the early diastolic dysfunction observed in the global PAD2^−/− model cannot be teased apart using this approach. In addition, the indirect impact on other cell types and tissues that express PAD2 may also influence the observed responses. It will therefore be crucial to investigate the role of PAD2 in age-related diastolic dysfunction using a tissue- and cell-specific model, such as a cardiomyocyte-specific PAD2^−/− model.

Although there is much more left to learn, the study by Shorthill et al⁵., has laid the groundwork for future investigations examining the role of PAD2-mediated citrullination in the aged heart and may lead to future insights regarding sex-dependent differences in the development of diastolic dysfunction. To advance knowledge of the importance of PAD2 and citrullination in the aged female heart, it will be essential to determine what regulates PAD2 expression in the heart, examine possible non-canonical roles of PAD2, and to tease apart the functional consequences of the reduction in protein citrullination on cardiac function. Should PAD2 prove to be a key regulator of these changes, this could also lead to the development of sex-specific therapeutic strategies targeting PAD2 and citrullination for age-related changes in cardiac function.