**3. Results**

#### *3.1. Successful Overexpression of Parkin in Skeletal Muscles of Sham and CLP Operated Mice*

Four weeks after the intramuscular injections of AAVs, mice were subjected to cecal ligation and perforation (CLP) to induced polymicrobial sepsis. Sham-operated mice were used as control. At baseline (prior to sham and CLP procedures), body weight values were similar in the sham and CLP groups, as shown in Figure 1A. Body weight loss was more pronounced in the CLP group relative to the sham group (−13.8 ± 1.4% vs. −4.7±1.1%, respectively, *p* < 0.05), as shown in Figure 1B. As shown in Figure 1C,D, the intramuscular injection of AAV-Parkin significantly increased *Park2* mRNA expression and Parkin protein content, in the skeletal muscles of both Sham-operated and CLP mice. These results demonstrate that our approach was successful in overexpressing Parkin in mouse skeletal muscle.

**Figure 1.** Effective Parkin overexpression in skeletal muscles of Sham and CLP operated mice. (**A**) Initial body weight and (**B**) percent of body weight loss in Sham-operated and or CLP mice. (**C**) qPCR analysis of *Park2* expression levels in the gastrocnemius muscles injected with either AAV-GFP or AAV-Parkin in Sham and CLP mice. (**D**) Representative Parkin immunoblots and its corresponding ponceau S stain performed on gastrocnemius samples of Sham and CLP mice injected with either AAV-GFP or AAV-Parkin. 1 = Sham-AAV-GFP; 2 = Sham-AAV-Parkin; 3 = CLP-AAV-GFP; 4 = CLP-AAV-Parkin. Data are presented as mean ± SEM (*n* = 7–9/group; \* = statistically significant; ns = not statistically significant).

#### *3.2. Parkin Overexpression Attenuates Sepsis-Induced Skeletal Muscle Atrophy*

The effect of Parkin overexpression on muscle fiber size was evaluated 48 h after CLP, based on our previous reports which revealed that limb muscle atrophy develops at this time point [30,33]. In the sham group, Parkin expressing muscles had larger myofiber diameters relative to those expressing GFP, as shown in Figure 2B,C. This observation is in line with our previous report [32]. In the CLP group, GFP expressing muscles displayed a trend towards smaller myofiber diameters and a decreased proportion of large fibers relative to those expressing GFP in the sham group, as shown in Figure 2B,D, all of which are indicative of myofiber atrophy. As shown Figure 2B,E, no sign of atrophy was detected in the Parkin overexpressing muscles of CLP mice when compared to the Parkin expressing muscles of Sham-operated mice. In addition, the Parkin overexpressing muscles of CLP mice displayed larger myofibers vs. the GFP expressing muscles of CLP mice, as shown in Figure 2B,F. These results indicate that Parkin overexpression prevented the development of muscle atrophy in the CLP group and increased muscle fiber diameter in the sham group.

**Figure 2.** The impact of Parkin overexpression and sepsis on skeletal muscle fiber size. (**A**) Representative gastrocnemius (GAS) cryosections stained for laminin in all experimental groups. Scale bar: 50μm. (**B**) Quantification of minimum Ferret diameter of GAS myofibers of Sham and CLP animals injected with either AAV-GFP or AAV-Parkin. (**C**) Minimum Ferret distribution of the GAS myofibers of Sham AAV-GFP (*n* = 8 mice; 316 ± 21 fibers per GAS were traced) vs. Sham AAV-Parkin (*n* = 8 mice; 300 ± 5 fibers per GAS were traced). (**D**) Minimum Ferret distribution of the GAS myofibers of Sham AAV-GFP (*n* = 8 mice; 316 ± 21 fibers per GAS were traced) vs. CLP AAV-GFP (*n* = 6 mice; 345 ± fibers per GAS were traced). (**E**) Minimum Ferret distribution of the GAS myofibers of Sham AAV-Parkin (*n* = 8 mice; 300 ± 5 fibers per GAS were traced) vs. CLP AAV-Parkin (*n* = 6 mice; 304 ± 18 fibers per GAS were traced). (**F**) Minimum Ferret distribution of the GAS myofibers of CLP AAV-GFP (*n* = 6 mice; 345 ± fibers per GAS were traced) vs. CLP AAV-Parkin (*n* = 6 mice; 304 ± 18 fibers per GAS were traced). Data are presented as mean ± SEM. (*n* = 6–8/group; \* = statistically significant).

#### *3.3. The Impact of Parkin Overexpression and Sepsis on Skeletal Muscle Catabolic Signaling*

We then investigated whether Parkin overexpression and sepsis affect the expression levels of apoptotic and autophagy-related genes. Neither Parkin overexpression nor sepsis affected the mRNA levels of pro-apoptotic *Bax*, *Bid*, *Bim*, and anti-apoptotic *Bcl2*, depicted in Figure 3A. As shown in Figure 3A, the expression level of BclXL was higher in septic animals. qPCR analyses revealed a significant increase in the mRNA levels of *Lc3b*, *Sqstm1*, *Gabarapl* and *Bnip3* in septic mice, shown in Figure 3B. The expression of *Gabarapl* was significantly higher in the Parkin overexpressing muscles of septic animals. No other impacts of Parkin overexpression on apoptotic and autophagy-related genes were observed. In line with our gene expression data, the protein contents of SQSTM1 (also known as p62) and BNIP3, two proteins regulating autophagy and mitophagy, were increased in septic animals, as shown in Figure 3C–E. The ratio of LC3-II to LC3-I was significantly increased in septic mice, suggesting an induction of autophagy, shown in Figure 3F. No impact of Parkin overexpression on the content of SQSTM1 and BNIP3 and the LC3-II to LC3-I ratio could be evidenced. We then assessed the expression levels of two key E3 ligases known to contribute to skeletal muscle atrophy [38,39], Fbxo32 (Atrogin-1) and Trim63 (MuRF1). The expression of these two E3 ligases was significantly increased in the skeletal muscle of septic animals, shown in Figure 3G. Parkin overexpression did not impact Fbxo32 and Trim63 expression. It is worth mentioning that neither Parkin overexpression nor sepsis had an impact on the content or phosphorylation levels of AKT and 4EBP1, two key proteins involved in the regulation of protein synthesis, as shown in Supplementary Figure S2. Taken altogether, these data indicate that Parkin overexpression did not attenuate sepsis-induced increases in catabolic signaling.

**Figure 3.** The impact of Parkin overexpression and sepsis on skeletal muscle catabolic signaling. (**A**) qPCR analysis of the mRNA expression of genes regulating apoptosis in the gastrocnemius (GAS) muscles of Sham and CLP animals injected with either AAV-GFP or AAV-Parkin. (**B**) qPCR analysis of autophagy-related gene expression in the gastrocnemius (GAS) muscles of Sham and CLP animals injected with either AAV-GFP or AAV-Parkin. *Gaba.* refers to *Gabarapl1*. (**C**) Immunoblot detection of SQSMT1(p62), BNIP3, LC3I/LC3II and GAPDH. (**D**) Quantification of SQSMT1 (p62) content. (**E**) Quantification of BNIP3 protein content. (**F**) Quantification of LC3I and LC3II protein content, as well as the LC3II to LC3I ratio. (**G**) qPCR analysis of *Fbxo32* (Atrogin-1) and *Trim63* (MuRF1) gene expression levels in the GAS muscles of Sham and CLP animals injected with either AAV-GFP or AAV-Parkin. 1 = Sham-AAV-GFP; 2 = Sham-AAV-Parkin; 3 = CLP-AAV-GFP; 4 = CLP-AAV-Parkin. Data are presented as mean ± SEM. (*n* = 6–9/group, \* = statistically significant; ns = not statistically significant).
