An association study of cyclase‐associated protein 2 and frailty

Abstract Frailty is a geriatric syndrome that results from multisystem impairment caused by age‐associated accumulation of deficits. The frailty index is used to define the level of frailty. Several studies have searched for molecular biomarkers associated with frailty, to meet the needs for personalized care. Cyclase‐associated protein 2 (CAP2) is a multifunctional actin‐binding protein involved in various physiological and pathological processes, that might reflect frailty's intrinsic complexity. This study aimed to investigate the association between frailty index and circulating CAP2 concentration in 467 community‐dwelling older adults (median age: 79; range: 65–92 years) from Milan, Italy. The selected robust regression model showed that circulating CAP2 concentration was not associated with chronological age, as well as sex and education. However, circulating CAP2 concentration was significantly and inversely associated with the frailty index: a 0.1‐unit increase in frailty index leads to ~0.5‐point mean decrease in CAP2 concentration. Furthermore, mean CAP2 concentration was significantly lower in frail participants (i.e., frailty index ≥0.25) than in non‐frail participants. This study shows the association between serum CAP2 concentration and frailty status for the first time, highlighting the potential of CAP2 as a biomarker for age‐associated accumulation of deficits.

gate the association between frailty index and circulating CAP2 concentration in 467 community-dwelling older adults (median age: 79; range: 65-92 years) from Milan, Italy. The selected robust regression model showed that circulating CAP2 concentration was not associated with chronological age, as well as sex and education. However, circulating CAP2 concentration was significantly and inversely associated with the frailty index: a 0.1-unit increase in frailty index leads to ~0.5-point mean decrease in CAP2 concentration. Furthermore, mean CAP2 concentration was significantly lower in frail participants (i.e., frailty index ≥0.25) than in non-frail participants. This study shows the association between serum CAP2 concentration and frailty status for the first time, highlighting the potential of CAP2 as a biomarker for age-associated accumulation of deficits.

K E Y W O R D S
aging, biomarker, cyclase-associated protein 2, frailty index Frailty is a clinically recognized condition characterized by increased vulnerability to stressors due to reduced homeostatic reserve (Clegg et al., 2013). From a biological perspective, frailty is driven by the lifelong accumulation of molecular and cellular deficits that involves different organs (e.g., skeletal muscle and brain) and systems (e.g., respiratory, cardiovascular, and endocrine). Frailty is indicated as the best approach to capture individuals' physiological decline and biological aging (Mitnitski et al., 2001). While there is a general agreement on frailty's theoretical definition, its clinical identification is challenging due to pathophysiological complexities and clinical manifestations . Moreover, multiple frailty definitions exist , and many operational approaches have been proposed over time (Morley et al., 2013). Frailty is most frequently assessed either using the phenotypic model or measuring the accumulation of health deficits through the frailty index (FI). While the frailty phenotype is focused on the physical domain of frailty (Cesari et al., 2014), the FI mirrors the individual's biological age and is expressed by the ratio of an individual's health deficits over the total possible deficits, evaluated within a comprehensive geriatric assessment (Mitnitski et al., 2001).
Several studies searched for frailty biomarkers to improve the understanding of its biological background and potentially support the development of specific interventions. The most studied circulating biomarkers are those related to the inflammatory response (Cannizzo et al., 2011). However, frailty's complex underlying pathophysiology makes identifying specific biomarkers extremely challenging. Most of the proposed frailty biomarkers have been investigated using the phenotypic model, approaching the condition as a physical syndrome. Exploring frailty's biological background, as measured by FI, might provide more information on the multidimensional aging phenomenon by including symptoms, signs, functional impairments, diseases, and laboratory abnormalities.
In this context, we focused on the cyclase-associated protein 2 (CAP2), an actin-binding protein that controls actin cytoskeleton dynamics and, thereby, regulates cellular processes such as muscle contraction and changes in synaptic morphology that underlie memory and learning . Remarkably, CAP2 expression is restricted to a limited number of tissues, including heart, skeletal muscle, and brain (Bertling et al., 2004), which are the organs mostly affected by frailty. CAP2 has been identified as a crucial regulator of the physiological functions of these organs and alterations in CAP2 have been associated to many age-related diseases, such as Alzheimer's disease (AD) (Rust & Marcello, 2022). For instance, alterations in CAP2 pathway can contribute to synaptic dysfunction and cognitive deficits in AD patients  and, thereby, CAP2 levels might be related to the neurocognitive component of frailty. Moreover, the relevance of CAP2 in controlling heart and skeletal muscle function has been observed in CAP2 knockout mouse (Field et al., 2015;Kepser et al., 2019) and confirmed by several human genetic studies (Aspit et al., 2019;Gurunathan et al., 2022), suggesting that CAP2 can be associated to sarcopenia, the biological substrate of physical frailty.
Given CAP2 biological functions, we measured its circulating concentration to assess its potential association with frailty, defined by the FI, in Italian older adults.
Participants' median age was 79 years (IQR: 75-83; maximum value = 92). Among participants, 55.7% were women, and 51.8% reported nine or more years of education. Their median FI was 0.22 (IQR: 0.15-0.29; maximum value = 0.57). Finally, their median CAP2 concentration was 5.21 ng/mL (IQR: 3.32-8.26; maximum value = 57.8) ( Table S1). No significant differences were found in the percentages of participants with or without the single health deficits included in the FI calculation, according to CAP2 concentration being below or equal to/above its median value (Table S2).
TA B L E 1 β coefficients (estimate), standard errors (SE), and p-values (p, from t test testing the null hypothesis that each parameter was equal to 0), as obtained from the selected robust linear regression models assessing the relationship between confounding factors and frailty index (FI) (independent variables) and cyclase-associated protein 2 (CAP2) concentration (dependent variable).  Our results support the hypothesis that circulating CAP2 concentration provides a snapshot of frailty. Although the FI provides a multidimensional measure of clinical phenotypes related to deficits, biomarkers such as CAP2 may assist in better targeting at-risk subpopulations, thus (i) ameliorating planning and implementation of personalized preventive and therapeutic interventions and (ii) supporting a more accurate prediction of frailty's trajectory. In addition, several studies have suggested that the age-associated decline in adaptive homeostasis is a major risk factor for many age-associated diseases, such as cancer, cardiovascular disease, and AD (Cannizzo et al., 2011). Therefore, CAP2 as a biomarker of frailty could be used to filter participants recruited for clinical trials, maximizing the intervention's effect size by selecting individuals particularly at risk.
The strengths of our analysis are the multidimensional assessment of older adults and the use of robust regression models to F I G U R E 1 Estimated marginal means of cyclase-associated protein 2 (CAP2) levels as derived from the robust linear regression model assessing the relationship between confounding factors and frailty index (FI) (dichotomous variable) and CAP2 concentration. Frail subjects were defined as those with a FI ≥0.25. 1 | E XPERIMENTAL PROCEDURE S

| Study design
At the geriatric outpatient clinic, Fondazione IRCCS Ca′ Granda Ospedale Maggiore Policlinico, Milan, Italy, 467 communitydwelling older adults underwent a comprehensive geriatric assessment and provided biological specimens between 2005 and 2020 (Appendix S1 for additional details). A 46-item FI was defined (Table S3) based on the geriatric evaluation, to include multimorbidity and disability phenomena, according to standardization criteria (Searle et al., 2008).

| CAP2 measurement
Serum CAP2 concentration was determined using a commercially available ELISA kit (n. IK5163; Immunological Sciences).

| Statistical analysis
Violations of the standard ordinary least squares assumptions sug- emmeans (Lenth, 2021). All authors read and approved the final manuscript.

ACK N OWLED G M ENTS
We thank Elisa Zianni and Annalisa Longhi for technical assistance and Evelyn Ferri for excellent practical work. The authors acknowledge the support of the APC central fund of the University of Milan. to EM, MUR Progetto Eccellenza).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article and its supplementary information files. The dataset analyzed during the current study are available from the corresponding authors on reasonable request.

E TH I C S S TATEM ENT
All participants provided informed consent to use their clinical and biological data for the research (study protocol number 0001669, January 16, 2020).