**1. Introduction**

Advancing age is associated with declining muscle mass, function, and strength, a condition referred to as sarcopenia which increases the risk of incurring negative health-related outcomes (e.g., disability, loss of independence, institutionalization, death) [1]. Hence, sarcopenia and its clinical correlates are major public health priorities. Physical activity, nutritional interventions, and multi-component programs have proven to be valuable strategies for managing sarcopenia [2–4]. Yet, no effective pharmacological treatments are currently available to prevent, delay, or treat sarcopenia, which is mostly due to the incomplete knowledge of the underlying pathophysiology [2]. To further complicate the matter, at the clinical level, sarcopenia shows remarkable overlap with frailty, a "multidimensional syndrome characterized by a decrease in physiological reserve and reduced resistance to stressors", often envisioned as a pre-disability condition [5]. Hence, the two conditions have been merged into a new entity, referred to as physical frailty and sarcopenia (PF&S) [6].

Mitochondrial dysfunction and sterile inflammation are invoked among the pathogenic factors of PF&S [7,8]. Derangements at different levels of the mitochondrial quality control (MQC) machinery have been reported in older adults with PF&S [7]. However, whether and how cell-based alterations may spread at the systemic level and impact muscle homeostasis is presently unknown.

One of the mechanisms by which cells communicate with each other involves a conserved delivery system based on the generation and release of extracellular vesicles (EVs) [9]. These vesicles transfer information between cells through several categories of cargo-enriched biomolecules (i.e., proteins, lipids, nucleic acids, and sugars), each of them selectively influencing different cellular domains [10]. This shuttle system also contributes to degradative pathways responsible for eliminating oxidized cell components, including mitochondria, by establishing inter-organelle contact sites [11]. In particular, in the setting of incomplete mitochondrial depolarization, cells may either delay autophagy to remove mildly damaged organelles or shift from mitophagy to the extrusion of mitochondrial components within EVs [12,13]. As such, the generation and release of mitochondrial-derived vesicles (MDVs) may represent a complement to MQC systems before whole-sale organelle is triggered [13,14].

Cell-free mitochondrial DNA (mtDNA) has been identified among the molecules released within exosomes that may act as damage-associated molecular patterns (DAMPs) [15]. One of the biological roles for these molecules is the activation of innate immunity through binding of their hypomethylated CpG motifs, resembling those of bacterial DNA, to membrane- or cytoplasmic-pattern recognition receptors (PRRs), including Toll-like receptor (TLR), nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) [16], and cytosolic cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) DNA sensing system-mediated pathways [17]. However, mtDNA is not the only mitochondrial constituent that may be displaced via MDVs and trigger these responses. Recently, the extrusion of mitochondrial components other than mtDNA has been reported within small EVs (sEVs) purified from the serum of older adults with Parkinson's disease (PD) [14]. However, whether and how this mechanism is in place in the setting of PF&S is unexplored.

In the present study, we purified sEVs from older adults with and without PF&S, quantified their amount, and characterized their content for the presence of mitochondrial components. The identification of specific derangements in sEVs in PF&S may shed light on its pathophysiology as well as suggest new biomarkers and possible biological targets for drug development.

#### **2. Materials and Methods**

#### *2.1. Participants*

Older adults aged 70+ with and without PF&S were recruited among the participants of the "BIOmarkers associated with Sarcopenia and Physical frailty in EldeRly pErsons" (BIOSPHERE) study [18]. BIOSPHERE was designed to determine and validate a panel of PF&S biomarkers through multivariate statistical modeling of biomolecules pertaining to inflammation, redox homeostasis, amino acid metabolism, neuromuscular junction dysfunction, and muscle remodeling pathways [18–20].

The operational definition used in the "Sarcopenia and Physical fRailty IN older people: multi-componenT Treatment strategies" (SPRINTT) project [21,22] was applied to diagnose PF&S: (a) physical frailty, based on a summary score on the Short Physical Performance Battery (SPPB) [23] between 3 and 9, (b) low appendicular muscle mass (aLM), according to the cut-points proposed by the Foundation for the National Institutes of Health (FNIH) sarcopenia project [24], and (c) absence of mobility disability (i.e., inability to complete the 400-m walk test) [25]. The present investigation involved a convenience sample of 21 participants, 11 older adults with PF&S and 10 non-sarcopenic non-frail (non-PF&S) controls. Participants were randomly chosen from the cohort of the BIOSPHERE study [18], among those from whom serum was available for vesicle purification.

The study was approved by the Ethics Committee of the Università Cattolica del Sacro Cuore (Rome, Italy; protocol number BIOSPHERE: 8498/15) and all participants signed an informed consent prior to inclusion. Study procedures and criteria for participant selection were described thoroughly elsewhere [18].

#### *2.2. Measurement of Appendicular Lean Mass by Dual X-Ray Absorptiometry*

Appendicular lean mass was quantified through whole-body Dual X-Ray Absorptiometry (DXA) scans on a Hologic Discovery A densitometer (Hologic, Inc., Bedford, MA, USA) according to the manufacturer's procedures. Criteria for low aLM were as follows: (a) aLM to body mass index (BMI) ratio (aLMBMI) < 0.789 in men and <0.512 in women, or (b) crude aLM < 19.75 kg in men and <15.02 kg in women [24].
