**1. Introduction**

Sepsis is a complex syndrome characterized by an overwhelming infection that results in a severe systemic inflammatory response. Sepsis causes diverse vascular, metabolic and endocrine abnormalities that lead to multiple organ failure, and often result in death [1]. Amongst the very deleterious effects of sepsis is severe weakness, which involves both respiratory and limb skeletal muscles [2–5]. In the short term, sepsis-induced respiratory muscle weakness leads to difficulty removing patients from mechanical ventilation, increases the risk of the recurrence of respiratory failure, prolonged hospitalization and increased mortality [6]. In sepsis survivors discharged from the intensive care unit, the long-term ramifications of sepsis-induced limb muscle weakness included functional impairment, limited physical activity and poor quality of life [7].

There is currently a lack of effective therapies to either prevent or treat sepsis-induced skeletal muscle weakness, due in large part to the fact that its molecular and cellular bases are poorly understood. However, one clue lies at the ultrastructural level, where significant accumulations of damaged and dysfunctional mitochondria are characteristic of sepsis-induced muscle dysfunction [8,9]. Indeed, Bready et al. showed that, in human skeletal muscle, sepsis results in decreased complex I activity (a key enzyme of the mitochondrial electron transfer system) and declined the ATP/ADP ratio in skeletal muscles [10]. These defects in muscle bioenergetics were also observed in a rat model of sepsis [11]. By studying biopsies obtained from septic patients, Fredriksson K et al. described a 30% decrease in complex IV activity in limb skeletal muscles [12]. Several studies on experimental animals also reported that sepsis results in decreased mitochondrial respiration [13–16] and an increase in the mitochondrial production of reactive oxygen species (ROS) in skeletal muscle [17,18]. Sepsis has also been shown to increase the levels of morphologically abnormal mitochondria, such as those with disorganized cristae, translucent vacuoles and even myelin-like structures [13,19–21]. Recently, Owen et al. showed that persistent muscle weakness in mice that have survived sepsis is associated with abnormal mitochondrial ultrastructure, decreased respiration, decreased activity of complexes of the mitochondrial electron transfer system and persistent oxidative damage to muscle proteins [21].

In healthy muscles, damaged or dysfunctional mitochondria are selectively recycled in a process, known as mitophagy (selective autophagy of mitochondria), which is primarily regulated through the PINK1-Parkin pathway. Parkin, an E3 ubiquitin ligase encoded by the *Park2* gene, is a 465 amino acid protein that translocates to depolarized mitochondria to initiate mitophagy. Parkin-dependent mitophagy is regulated by PTEN-induced kinase 1 (PINK1), which acts upstream from Parkin. In healthy mitochondria, PINK1 is imported into the inner mitochondrial membrane and cleaved by PARL [22]. Cleaved PINK1 is then released into the cytosol where it is degraded by the proteasome system. In depolarized mitochondria, the importation of PINK1 into the inner mitochondrial membrane is blocked. PINK1 is no longer degraded and becomes phosphorylated and stabilized on the outer mitochondrial membrane [23–26]. Phosphorylated PINK1 triggers the recruitment of Parkin to the mitochondria. Parkin then ubiquitinates outer mitochondrial membrane proteins, including the fusion proteins MFN1, MFN2, MIRO and TOMM20 [27]. The degradation of MFN1 and MFN2 triggers mitochondrial fission and fragmentation, both of which are important to the recycling of mitochondria by the mitophagy pathway [28]. The functional importance of the PINK1-Parkin mitophagy pathway in regulating skeletal muscle mitochondrial function and quality in sepsis remains unknown. Recently, we reported that the genetic deletion of Parkin leads to the poor recovery of cardiac function in septic mice and increased sepsis-induced mitochondrial dysfunction in the heart [29]. We also demonstrated that autophagy is significantly induced in the skeletal muscles of septic mice and that the induction of autophagy is associated with increased muscle Parkin levels, suggesting that mitophagy was induced [20,30]. However, several morphologically and functionally abnormal mitochondria were observed in the electron micrographs of septic muscles, indicating that the mitophagy that was induced was likely insufficient to the task of completely recycling defective mitochondria [20,30]. Based on this reasoning, we hypothesized that enhancing mitophagy through Parkin overexpression would attenuate the impact of sepsis on skeletal muscles and their mitochondria. To test this hypothesis, Parkin was overexpressed for four weeks in the skeletal muscles of young mice using intramuscular injections of adeno-associated viruses (AAVs). The cecal ligation and perforation (CLP) procedure, a widely used model of sepsis [31], was used to induce sepsis. Sham-operated animals served as controls. We found that Parkin overexpression prevents sepsis-induced mitochondrial morphological injury and reverses

the decline in mitochondrial protein content. We also found that Parkin overexpression protects against sepsis-induced myofiber atrophy. These findings indicate that defective mitophagy in sepsis can be therapeutically manipulated as a means of counteracting sepsis-induced muscle dysfunction.
